JP2014039562A - Method for rapid multiplexed amplification of target nucleic acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for rapidly amplifying single or multiple gene loci in a nucleic acid sample, a thermal cycler, and a useful system to carry out this method.SOLUTION: A rapid and multiplex PCR system capable of rapidly forming an amplified nucleic acid product (for example, complete STR profile) from target nucleic acid is provided. A multiplex PCR system equipped with a micro fluid biochip and a special thermal cycler is provided. Obtained STR profile can satisfy a forensic guideline which is related to signal strength, inter-loci peak height balance, heterozygous peak height ratio, incomplete non-template nucleotide addition, and stutter.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS
This application is based on 35 USC §119 (e), U.S. Provisional Application No. 60 / 921,802 (filed; April 4, 2007), U.S. Provisional Application No. 60 / 964,502 (filed; 2007 8 month 13 day), and, U.S. provisional application No. 61 / 028,073 (filed; claims the benefit of the filing date of Feb. 12, 2008). Each of these is by reference hereby in their entirety are incorporated herein. This application is further, by reference to the two US patent applications filed on the same date as this application, incorporated in its entirety. One of these is a "INTEGRATED NUCLEIC ACID ANALYSIS (integrated analysis of nucleic acids)," and entitled Application (Attorney Docket No. 07-801-US), the other one, "PLASTIC Microfluidic SEPARATION it is the aND dETECTION pLATFORMS (separation platform relates to a microfluidic plastic and detection) "and entitled application (Attorney Docket No. 07-865-US).

The present invention generally provides methods for rapidly amplifying one or more loci in a nucleic acid sample, a thermal cycler, and to a system useful for performing this method.

Polymerase chain reaction (PCR) is an enzyme reaction that promotes amplification of exponentially rapid nucleic acid sequence in vitro (in vitro). In forensics, based on the amplification of small regions of the human genome has a class of repetitive DNA, known as tandem repeats (STR), to identify the individual, can be utilized PCR is there. Unit length in a particular STR are repeated in the range between 2 and 10 base pairs. Moreover, STR is generally, although included in the flanking non-coding sequences may also be included in the coding region (Edwards, et al., Am. J. Hum. Genet. 1991, 49, 746 -756). Within the human genome, there are hundreds of thousands of STR loci, they are average and occur every 6~10kb by (Beckman and Weber, Genomics 1992,12,627-631), further, a wider variety of forms going on as to also visible (Edwards et al., Trans. Assoc. Am. Physicians 1989, 102, 185-194). STR analysis has become a major tool in the forensic equipment, the series of applications (inspection of being a father, the person identified in the population disaster, and, including the routine typing of children) is increasing.

For the synthesis of PCR products having the desired high specificity, a plurality of commercially available STR kits have been developed. However, the current STR technique, there is a very large area capable of improving. Most importantly, the average time to complete the multiplex PCR using the commercially available STR typing kit is approximately 2.14 hours. Time-consuming and labor intensive in these analyzes, have contributed to the backlog in forensic laboratories. Birth of automated instrumentation for processing multiple samples simultaneously, while has helped to alleviate the serious bottleneck in typing throughput, the number of samples to be analyzed is increased, the process further the You should request the acceleration. Furthermore, the need to increase the sensitivity of the STR analysis, there is also a need to improve the detection of amplified product (Gill, Croat. Med. J. 2001, 42, 229-32). Useful STR kits at present includes a 9-16 loci, work is underway in the art to increase the number of possible loci detecting. The specific application of STR analysis in this field, can be implemented by using four or more loci.

Further, PCR, and it can also be applied in a wide range of clinical situations. Eg, PCR, and it is also possible to use Streptococcus group A, Staphylococcus aureus methicillin-resistant, in the diagnosis of bacterial infections, such as vancomycin-resistant enterococci. Further, PCR is generally compared to culture-based diagnostic techniques, have higher sensitivity. Similarly, it is also possible to diagnose a fungal infection. The PCR, respiratory viruses (e.g., respiratory syncytial virus, adenovirus, and influenza and parainfluenza viruses), genitourinary viruses (e.g., herpes virus and typing human papillomavirus), osteomyelitis (e.g. , herpes virus, Epstein-Barr virus, varicella zoster virus, and entero-viruses), and, hepatitis (e.g., can also be used in the diagnosis of hepatitis B and C). PCR is also useful in preimplantation genetic diagnosis (including diagnosis of aneuploidy assessment and genetic disorders). From oncology to rheumatology, and, from hematology to gastroenterology, finding areas of medicine which is not affected by PCR is difficult.

Further, PCR is different non-clinical situations (veterinary identification (similar to STR typing of human), veterinary diagnostics, food safety evaluation, detection of pathogens in agriculture, and, Famako It is applied also in including Genomics). Increasing importance to while some applications are those that are associated with a specific biological warfare agents in clinical and environmental samples. Each amount of product appearing in the subsequent reaction after amplification cycle allow quantification and real time PCR (as PCR related deep) are used in PCR itself essentially the same purpose (Espy et al., Clinical Microbiology Reviews 2006, 19, see 1656-256).

The most commercially useful temperature cycle instrument, it, from the block temperature as opposed ones with PCR solution temperature, that directly receives the temperature feedback, and that they, and controls the block temperature in point it is limited. As a result, (essential thing for the success of the PCR) temperature profile of solution, the desired profile is a possibility that drastically different.

Furthermore, most of the literature on the increase in speed and sensitivity of the PCR is focused on one specific locus amplification at a time ( "singlet Analysis"), further forensic STR typing, clinical such as those needed for diagnosis and non-clinical applications, for simultaneous amplification of multiple loci ( "multiplex analysis"), only limited success has been reported. For example, a chamber of 160nL which joins the integrated heater, four STR included in Y-STR analysis of 80 minutes (detection limit, 20 copies in the template DNA) to amplification and separation It has been shown to be capable of (Liu et al., Anal. Chem.2007, 79, 1881-1889). Also, the sensitivity of the PCR that has been increased by reducing the reaction volume of the PCR have been reported for PowerPlex (TM) 16 system. However, it attempts to increase the reaction rate is not made (Schmidt et al., Int. J. Legal Med. 2006, 120, 42-48). However, what is needed in the art, there are no reports give amplification time is significantly reduced. Hopwood et al., International Congress Series 1288 (2006) 639-641) have reported the amplification of 100 minutes using a set of eleven STR primers. For the clinical diagnosis, a panel of seven common respiratory virus, in PCR analysis that require inter-97.5 minutes, and is amplified using nano chip system (Takahashi et al., J. lin . Microbiol 2008, doi:.. 10 1128 / JCM 01947-07).

Forensic person identification by STR typing, clinical diagnosis, and such detection of biological warfare agents, many applications of PCR (and real-time PCR), is extremely time constraints. And, many applications are best performed in multiple situations. Furthermore, many of these applications, a limited sample (e.g., a small number of pathogens from clinical samples or environmental samples, or a small number of human cells from forensic samples) A ​​as obtained only situation the sensitivity of the reaction is used in situations where an important meaning.

Notably, Horsman et al, (J. Forensic Sci., 2007, 52, 784-799) is 792 Id of, "as evidenced by the large amount of literature on this topic, PCR is analytical micro between the tip researchers, general became studies. However, for forensic DNA analysis, many means have been left. commercially available forensic STR kit for development or further , using multiple STR amplification in a single device extensive work is not observed. However, when fully developed is the microchip PCR is undoubtedly, a large amount of time and cost in the forensic community states that will be saved to. Thus, for a wide range of applications, successfully co-amplification of multiple loci in a nucleic acid sample Hisage To, to the rapid and sensitive method, there is a need in this field.

Equipment of the present invention, biochips, methods and systems are based on the actual temperature at least partially solution, by monitoring and controlling the thermal cycler, rapidly, controllably and reproducibly, PCR solution the providing function of heating and cooling. Instrument of the present invention disclosed herein, the biochip, the method and system, by incorporating special thermo sensor not in commercially available thermal cycler, in order to avoid excessive heating (or insufficient heating), Bio the reaction temperature of the solution in the chip, provides the ability to monitor and / or control. Such temperature on the ability to rapidly heat and cool the reaction solution, it becomes possible to minimize the rise time and fall time, the majority of total step time, incubation time at the desired temperature it is possible to be. Furthermore, the device of the present invention which are provided herein, biochips, methods and systems, causes rapid change the temperature of the reaction solution, discloses the ability to balance the temperature. Thus, possible speed of the amplification reaction proceeds, is greatly increased.

Equipment of the present invention, by using a biochip, the method and system, amplification time of high-speed multiplex PCR (short as 17 minutes) is obtained. Based on the teachings of the present invention, it is possible to shorten further the time. Furthermore, rapid PCR methods of the present invention are effective over a wide dynamic range, are extremely sensitive, yet correspond to a wide range of commercially available enzymes and reagents. Respect forensic applications, devices, biochips, the method and system of the present invention, the time required to generate a complete profile satisfying interpretation guidelines STR analysis, it is possible to greatly reduce .

In a first aspect, the present invention provides a thermal cycler comprises a temperature control element (TCE), a first surface of the TCE is holding the sample chamber and thermosensor holding the solution detection chamber is configured to receive, thermo sensor, in order to set or keep the samples on the desired temperature, giving feedback to the TCE, to provide a thermal cycler. In a second aspect, the present invention provides a thermal cycler which is arranged to monitor the temperature of the first surface of the TCE, further comprising a second thermosensor.

In a second aspect, the present invention is the system having the biochip, the biochip comprises one or more reaction chambers has a portion of the biochip having a volume (volume), each reaction chamber further comprises an outlet channel of the inlet channel and the microfluidic microfluidic, each reaction chamber, a system that is less than 200μm from the contact surface of the biochip substrate, further, the thermal It includes a cycler, the thermal cycler is provided with temperature control elements (TCE), the first surface of the TCE, is configured to receive and have the substrate and thermosensor holding samples the thermo sensor, as well as measuring the temperature of the sample in the substrate, the desired temperature Sample are arranged to provide feedback to the TCE to set or maintain, the thermal cycler, to provide a system, a in contact surface and the heat conduction of the biochip substrate.

In a third aspect, the present invention is the system having the biochip and a thermal cycler, the biochip comprises one or more reaction chambers, each reaction chamber, a portion of the biochip having a volume includes a further, has the outflow channel of the inlet channel and the microfluidic microfluidic, each reaction chamber, which is less than 100μm from the contact surface of the biochip substrate, further, the thermal cycler, temperature and a control element (TCE), the first surface of the TCE, sample is configured to receive and have the substrate and thermosensor holds, the thermo sensor, temperature of the sample in the substrate as well as measure, in order to set or keep the samples on the desired temperature CE is arranged to provide feedback to the thermal cycler, to provide a system, a in contact surface and the heat conduction of the biochip substrate.

In a fourth aspect, the present invention provides a method for amplifying a plurality of loci in a nucleic acid solution simultaneously, includes a step of providing one or more reaction chambers, each reaction chamber, (i) amplified nucleic acid solution containing at least one copy of at least one target nucleic acid to be, (ii) one or more buffers, (iii) one or more salts, (iv) a plurality of loci to be amplified the corresponding set of primers respectively, (v) a nucleic acid polymerase, and, (vi) a method has nucleotides, and the temperature of the nucleic acid solution in the reaction chamber, degenerative conditions, annealing conditions and extension between the state, for a predetermined number of cycles, at a heating and cooling rate of about four to one hundred and fifty ° C. / sec, continuously thermally Is ring, thereby, within about 97 minutes, to provide a method, which includes the step, the generating a plurality of amplified loci to each reaction chamber.

In a fifth aspect, the present invention provides a method for amplifying a plurality of loci in a nucleic acid solution simultaneously, includes a step of providing one or more reaction chambers, each reaction chamber, (i) amplified nucleic acid solution containing at least one copy of at least one target nucleic acid to be, (ii) one or more buffers, (iii) one or more salts, (iv) a plurality of loci to be amplified the corresponding set of primers respectively, (v) a nucleic acid polymerase, and over (vi) a method has nucleotides, and the temperature of the nucleic acid solution in the reaction chamber, a predetermined number of cycles, about at 4 to 150 ° C. / sec heating and cooling rate, continuously thermally cycled, thereby, within about 97 minutes, the reaction Ji The method comprising the step, the generating a plurality of amplified loci members to provide.

In a sixth aspect, the present invention provides a method for simultaneously amplifying five or more loci in a nucleic acid solution includes a step of providing one or more reaction chambers, each reaction chamber, (i ) nucleic acid solution containing at least one copy of at least one target nucleic acid to be amplified, (ii) one or more buffers, (iii) one or more salts, five to be amplified (iv) set of primers corresponding to the above locus, (v) a nucleic acid polymerase, and a method having a, (vi) nucleotides, the temperature of the nucleic acid solution in each reaction chamber, degenerative conditions, annealing conditions and between the extended state, for a predetermined number of cycles, at a heating and cooling rate of about four to one hundred fifty ° C. / sec, continuously thermally 循 Is allowed, thereby providing a method, which includes the step, the generating five or more amplified loci to each reaction chamber.

In a seventh aspect, the present invention provides the integrated bio-chip system has a biochip comprising at least two reaction chambers are in fluid micro fluid communication, the first reaction chamber, a thermal cycler and has thermal conductivity, the thermal cycler is provided with a temperature control element (TCE), the first surface of the TCE, configured to receive a substrate and thermosensor holding the sample cage, the thermo sensor, as well as measuring the temperature of the sample in the substrate is arranged to provide feedback to the TCE to set or maintain the sample to the desired temperature, the contact surface of the biochip, and first surface and the thermal conduction in a thermal cycler, further thermal cycler, A second reaction chamber connected one reaction chamber and fluid, nucleic acid extraction, nucleic acid purification, prior to nucleic acid purification PCR, purification after PCR, purification before sequencing, sequencing, sequencing purification after the nucleic acid isolation, nucleic acid detection, reverse transcription, purification prior to reverse transcription, after reverse transcription purification, nucleic ligation, nucleic hybrid or a second reaction chamber suitable for quantifying cage, the first reaction chamber, which is less than 200μm from the contact surface of the biochip, to provide an integrated biochip system.

Certain preferred embodiments of the present invention is, from the detailed description and the appended claims rather than later in certain preferred embodiments, will become apparent.

Is a photograph of the embodiment of the thermal cycler of the present invention. For use with a thermal cycler shown in Figure 1A, is a photograph showing an embodiment of the 16-lane microfluidic biochips. The standard STR cycling protocol (total cycle time: 145.1 min) as shown here about one thermal cycle in a graph showing the temperature profile of the block and the reaction solution. Fast cycle protocol (total cycle time: 19.56 min) shown here about one thermal cycle in a graph showing the temperature profile of the block and the reaction solution. Fast cycle conditions (total cycle time: 17.3 minutes) about one thermal cycle for the thermal cycler of the present invention utilizing the a graph showing the temperature profile of the heat pump and the reaction solution. Using the template DNA of 0.5ng produced in biochip reaction according to the present invention, it is a graph showing the STR profile. Using the template DNA of 0.5ng produced in the tube a reaction according to the present invention, is a graph showing the STR profile. For the signal strength in the biochip reaction is a graph showing the effect of DNA template level. For the signal strength in the tube reactions is a graph showing the effect of DNA template level. For Ratio (PHR) of the peak height of the heterozygotes in biochip reactions is a graph showing the effect of DNA template level. For PHR in tube reactions, it is a graph showing the effect of DNA template level. To the addition of the nucleotide which does not use the template in biochip reactions (NTA), it is a graph showing the effect of DNA template level. For NTA in tube reactions, is a graph showing the effect of DNA template level. For stutter in biochip reactions is a graph showing the effect of DNA template level. For stutter in tube reactions, it is a graph showing the effect of DNA template level. Ing template DNA was generated by a set of COfiler (TM) primer utilizing a graph showing the profile for biochip reaction (top) and tube reaction (bottom). Ing template DNA was generated by a set of Identifiler (TM) primer utilizing a graph showing the profile for biochip reaction (top) and tube reaction (bottom). As described in Example 5, it is a graph showing a profile of an embodiment of a sequencing reaction.

To obtain a high-speed multiplex nucleic acid amplification such as PCR, the present invention is capable of reaction conditions can be used to amplify multiple loci in target nucleic acid samples, the temperature cycling instrumentation, and the reaction vessel I will provide a. As indicated by the example shown, the speed of the temperature cycling method of the present invention, by utilizing the method described in a thermal cycler and individual invention, performed in a microfluidic biochip it is possible.

The method provided by the present invention, in addition to the application using a biochip and a thermal cycler described herein, is intended to enable rapid multiplex amplification in multiple applications. For example, conventional thermal cycler (e.g., block-based thermal cycler, and Roche LightCycler (TM)) using a thin tube in, and, amplification methods other than temperature cycles of PCR (e.g., isothermal PCR or rolling circle amplification) use, are specifically contemplated.

The method provided by the present invention, biochips, and thermal cycler, within a particular nucleic acid solution present in at least 0.006ng amount of human genomic DNA (approximate amount of DNA in a single nucleated human cells) , in less than 100 minutes, it is possible to amplify multiple loci. In other embodiments, the amplification is less than 90 minutes, less than 80 minutes, less than 70 minutes, less than 60 minutes, less than 50 minutes, less than 40 minutes, less than 30 minutes, less than 20 minutes, less than 17.7 minutes, 15 minutes less, resulting in less than less than 10 minutes, or 5 minutes

In another embodiment, bacterial, viral origin, fungal origin, in the genome of the animal-derived or plant, as beginning at least one copy of the locus or multiple loci of the singular in the nucleic acid target, the plurality of genes it is possible to amplify a seat. For example, the sample to be analyzed, prior to the multiplex amplification reaction, less than 1000 copies of the target nucleic acid, 400 fewer than copying, 200 fewer than copying, less than 100 copies, of less than 50 copies, 30 less than copying, less than 10 copies, or may include at least one copy. Furthermore, the target nucleic acid loci, if included in more than one in the copy in the genome, for amplification, it is also possible to use a single genome equivalent to DNA . When Generally, according to the methods described herein, in each of the target nucleic acid in the sample, at least two loci, and the loci of up to approximately 250, can be amplified simultaneously. Furthermore, according to the method described herein, the nucleic acid of the plurality of targets, at least two loci, and the loci of up to approximately 250, can be amplified simultaneously.

The target nucleic acid to be used here, may be any nucleic acid (e.g., a human nucleic acid, bacterial nucleic acids, or such nucleic acids of the virus). The target nucleic acid sample, e.g., one or more cells, tissues, or body fluids (e.g., blood, urine, semen, lymphatic fluid, cerebrospinal fluid, or amniotic fluid, etc.), or other biological samples (e.g. , tissue culture cells, buccal swabs, mouthwash, excreta, tissue slices, such as aspiration biopsy), and, archaeological samples (e.g., can be a nucleic acid sample from the bone or mummified tissue) it is. For example, a nucleic acid target, DNA, RNA or, may be a DNA product of RNA exposed to reverse transcription. For a sample of the target can be obtained from any source. As the source, for example, eukaryotes, plants, animals, vertebrates, fish, mammals, humans, other than humans, bacteria, microbes, viruses, biological sources, serum, plasma, blood, urine, semen, lymph, cerebrospinal fluid, amniotic fluid, biopsies, needle aspiration biopsies, cancer cells, tumors, tissues, cells, cell lysates, cell lysate untreated, tissue lysates, tissue culture cells, buccal swabs, mouthwash, excrement, mummified tissue, forensic sources, body anatomical, archaeological sources, infections, nosocomial infections, product source, drug formulations, biomolecules product, protein preparations, lipid formulations, carbohydrate formulation, inanimate, air, soil, sap, metal, fossil, roots cut soil, and / or, or extraterrestrial materials and sources on the other earth include (but not limited to). Similarly, sample may comprise a mixture of material from a single source or a plurality of different sources. For example, a nucleic acid of the bacterial infection or infection virus, can be amplified with human nucleic acid (when the nucleic acid from such infected cells or infected tissue is amplified using the methods disclosed) . Type of sample practical targets, eukaryotic samples, plant samples, animal samples, vertebrate samples, fish samples, mammalian samples, human samples, non-human samples, bacterial samples, microbial of the sample, a sample of the virus, biological samples, serum samples, plasma samples, blood samples, urine samples, semen sample, a sample of lymph, cerebral spinal fluid sample, a sample of amniotic fluid, a biopsy sample, sample needle aspiration biopsy, cancer cells of the sample, a tumor sample, a tissue sample, cell sample, a sample of cell lysate, a sample of cell lysate untreated sample of tissue lysate, a sample of tissue culture cells , oral swab sample, mouthwash sample, sample of excrement, was mummified Woven samples, body sample, archaeological samples, infection samples, samples of nosocomial infection, samples of the product, the drug formulation samples, samples of biomolecules product, protein formulation sample, the lipid formulation sample, samples of carbohydrate preparations, inanimate samples, air samples, soil samples, sap samples, metal samples, fossil samples, samples of roots cutting soil, and / or, are or extraterrestrial samples on another planet . Type of forensic samples, blood, dried blood, stains of blood, buccal swabs, fingerprints, touch the sample (for example, the edge of a drinking glass, the inside of the peripheral edge of the baseball cap, or, in cigarette butts remaining epithelial cells, etc. ), chewing gum, the contents of the stomach, is saliva, nail scrapings, soil, a sample of sexual assault, hair, bone, skin and solid tissue such as. Type of environment samples, swab samples from unfiltered (or being filtered) air and water, soil, surface, envelope, and powders, and the like.

For example, the method shown here, it is possible to provide a nucleic acid sample such, amplified as to produce the appropriate data (data in particular meet the guidelines forensic interpretation) for the analysis is forensic interpretation . Such guidelines, signal strength, balance in peak height in the interior loci, the ratio of the peak height at heterozygotes (PHR), the addition of incomplete nucleotide without using a template (NTA), and, stutter (Scientific Working Group on DNA Analysis Methods, Short Tandem Repeat (STR) Interpretation Guidelines. Forensic Science Communications, 2000, 2 (3)) are included.

Here the phrase being used "fluid communication (fluid Communication)", the "two chambers, or has other member or region containing a fluid connected to each other, thereby, these two chambers shows that it is possible to flow fluid between the member or region ". Thus, the two chambers are "fluid communication", for example, may be connected to each other by the microfluidic channel between the two chambers, thereby, it is made to flow freely fluid between the two chambers It can become. Such microfluidic channel is optionally may comprise a one or more valves therein. Then, in order to block the fluid communication between the chambers (for and / or control), it is possible to these valves closed closing ie.

The term is used herein, "poly (methyl methacrylate)" or "PMMA" refers to synthetic polymers methyl methacrylate. This, Plexiglas (TM), Limacryl (TM), R-Cast (TM), Perspex (TM), Plazcryl (TM), ACRYLEX (TM), ACRYLITE (TM), ACrylplast (TM), Altuglas (R), those sold under the trade names of Polycast (TM) and Lucite (TM), and U.S. Pat. No. 5,561,208, each of Nos. 5,462,995 and No. 5,334,424 (which is incorporated herein by reference it includes polymers described in by being) (but not limited to).

The term "polycarbonate" as used herein, polyesters of carbonic acid and glycol, or means a divalent phenol. Examples of such glycols or divalent phenols, p- xylylene glycol, 2,2-bis (4-hydroxyphenyl) propane, bis (4-oxyphenyl) methane, 1,1-bis (4- oxy) ethane 1,1-bis (oxyphenyl) butane, 1,1-bis (oxyphenyl) cyclohexane, 2,2-bis (oxyphenyl) butane, and mixtures thereof. Also, include those sold under the trade names of Caliber (TM), Makrolon (TM), PANLITE (trade mark), Makroclear (TM), Cyrolon (TM), Lexan (TM) and Tuffak (TM) (However, but are not limited to).

The term "nucleic acid" as used herein, single-stranded or double-stranded DNA and RNA, and an improved base, in an alternative nucleic acid containing sugars and backbone, any or all of the shape It is intended to encompass. Accordingly, the term "nucleic acid" may be single-stranded or double-stranded DNA and RNA (and, partially single-stranded or partially capable DNA and RNA shape of things to be double-stranded), cDNA , aptamers, peptide nucleic acid ( "PNA"), 2'-5 'DNA (synthetic material having a shortened backbone having compatible base interval a structure of DNA; 2'-5' DNA is usually, B shape and the DNA is never crossed readily crosses the RNA), and, it should be understood to include locked nucleic acid ( "LNA") (where not limited thereto is not). Nucleic acid derivative, the same or improved binding in pairing properties of a base, with a cross and includes a known derivatives of natural nucleotides. "Similar" shaped in purines and pyrimidines are known in the art, aziridinylcytosine, 4-acetylcytosine, 5-fluorouracil, 5-bromouracil, 5-carboxymethyl-aminomethyl-2-thiouracil, 5-carboxymethyl-aminomethyl uracil, inosine, ^{N 6} - isopentenyladenine, 1-methyl-adenine, 1-methyl pseudoephedrine uracil, 1-methylguanine, 1-methyl inosine, 2,2-dimethyl guanine, 2-methyl adenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, ^{N 6} - methyl adenine, 7-methylguanine, 5-methylaminomethyl uracil, 5-methoxy aminomethyl-2-thiouracil, beta -D- mannosyl queue o Shin , 5-Metokishiu Rashiru, 2-methylthio -N-6- isopentenyladenine, uracil-5-oxy acetic acid methyl ester, pseudoephedrine uracil, Kueoshin, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5- methyl uracil, uracil-5-oxy acetic acid and, contains a 2,6-diaminopurine (but not limited to). Derivatives of a DNA backbone provided by the present invention, phosphoric acid esters, phosphorothioate, dithiophosphoric acid, methylphosphonic acid, phosphoramidate, acrylic phosphate triester, sulfamate, 3'-thioacetal, methylene (methylimino group ), 3'-N-carbamate, morpholino carbamate, and peptide nucleic acid (PNA), methylphosphonate linkage or, alternative methylphosphonate, and phosphoric acid ester linkage (Strauss-Soukup, 1997, Biochemistry 36: 8692-8698), and include a benzyl phosphonic acid chain are described in U.S. Patent No. 6,664,057. It should be noted that, "OLIGONUCLEOTIDES AND ANALOGUES, A PRACTICAL APPROACH, F.Eckstein editing, IRL Press at Oxford University Press (1991)", "Antisense Strategies, Annals of the New York Academy of Sciences, Volume 600, Baserga and Denhardt, eds (NYAS 1992 ) "," Milligan, 1993, J. Med Chem 36:.. 1923-1937; Antisense Research and Applications (1993, CRC Press) ", see also. These nucleic acids, it is also possible to extract from the cells, can be formulated synthetically according to any means known to those skilled in the art. For example, these nucleic acids, chemically, from cDNA or mRNA that among other sources, can be synthesized or transcribed or reverse transcription.

The term used "via (Via)" herein means a through hole formed in the solid material. Thus, fluid communication between the top and bottom surfaces of this material is possible.

Here the term being used "loci (locus)" or "multiple loci (loci)" is used herein in one as defined or more nucleic acids (e.g., one or more chromosomes it means one or more specific locations in).

The term is used, "STR locus" and "multiple STR loci" Here, in a specific locus in the target nucleic acid, a repeating pattern of two or more nucleotides, is meant a nucleotide sequence . The repeating pattern, it is possible in the length range of the base pairs from 2 to 10 (bp), in general, in the non-coding intronic regions.

According to one aspect of the present invention, rapid, controllably, and with reproducible method, thermal cycler having the ability to heat and cool the reaction solution is provided. Example embodiments of the thermal cycler of the present invention is shown in Figure 1A. Functions for rapidly heating and cooling the temperature of the reaction solution, while allowing the rise time and fall time is minimized, in step the total time, dominance culture time at the desired temperature and It makes it possible to become. This makes it possible to minimize the multi-cycle time.

By utilizing a single or heat sink in fluid communication with the temperature control element (TCE), it is possible to obtain a high heating and cooling rates. The TCE, means for heating and cooling, thermo sensor, has a controller, and a power supply for receiving a signal from the thermo sensor. In a preferred embodiment, the first surface of the TCE, can be configured to receive a sample chamber. The sample chamber has a solution and detection chamber, the detection chamber has additional thermosensor. In this setting, it is disposed in the detection chamber in which the thermo sensor is attached to the TCE. As a result, it is, to simulate the status of the sample chamber. The detection chamber is made to have a sample chamber same laminated material as. Thermocouple mounted within the temperature sensor is in a position similar to the position of the sample in the sample chamber in this structure, are embedded. This sensor informs the effective temperature of the solution in the sample chamber. Commercially available types -T or Type K thermocouples (Omega Engineering (Stamford, CT) Ltd.) is the most appropriate. However, it is also possible to use thermocouple and thermosensor other types (including thermistors, semiconductor and infrared). Thermosensor in the sensing chamber provides a feedback to the TCE. Thus, the sample is set or maintained at a desired temperature. Thus, without inserting a thermosensor inside of the reaction chamber itself, the temperature of the sample becomes indirectly possible to measure and control. Alternatively, a thermosensor, and directly placed in a reaction chamber, it is possible to set or maintain the temperature of the sample. Thus, it is possible to eliminate the need for detection chamber. The skilled artisan will appreciate that, in accordance with the teachings of the present invention, it is also possible to use other types of sensors (e.g., pressure sensor).

The first surface of TCE, can be configured to receive the substrate substantially planar. This, for example, in order to receive a substrate (e.g. biochip (described later)), by forming a recess in the first surface. Alternatively, the TCE, it is also possible to configure so as to receive one or more thin-walled tubes. The thin-walled tube is defined as a tube having a thickness wall diameter smaller range than 200 [mu] m. The heat sink is preferably a high-performance heat sink. Examples of such a heat sink, for example, a fan-cooled heat sink (of the copper base with a cooling fan) and the like (but not limited to). More preferably, the heat sink has a thermal resistance of less than about 0.4 ° C. / W, can be made of copper base and fins of the fan-cooled. Specific non-limiting examples of high-performance heat sink, E1U-N7BCC-03-GP (Coolermaster, Taiwan ROC) is.

Thermal cycler of the present invention, further arranged to monitor the temperature of the first surface of the TCE, may comprise a thermo sensor. As desired, in order to further improve the temperature control of the sample, it is also possible to add additional thermosensor. Can be monitored auxiliary temperature, the temperature of the plurality of regions on the substrate (or within) the temperature of the plurality of areas on the heat sink (or within) the temperature of the cooling air in and out, of the sample in and out of temperature, and, the environmental temperature, and the like.

Good thermal conduction between the TCE and the heat sink is desired. If the two mating surfaces are properly arranged, close physical contact becomes sufficient to provide adequate heat transfer between these two members. To enhance the thermal coupling, it is possible to use a thermal interface material (TIM) between the TCE and the heat sink. Such TIM adhesives, greases, phase change material (PCM), metallic thermal interface materials, Serammikku Thermal Interface materials, soft metal alloys, indium, alumina nano-layer coating, submicron film, glycol, water, oil , antifreeze, epoxy compounds, and other include those (but not limited to). Specific examples are, Arctic Silver i.e. Ceramique (Arctic Silver, Visalia, CA ; 0.007 ℃ -in 2 / W compound having a smaller thermal resistance), compressible heat spring HSD4 (Indium Corp, Utica, NY ), HITHERM (GrafTech International Holdings Inc., Lakewood, OH), or the like direct binding of TCE to the surface of the heat sink. Greater than 2 psi, or greater than 5 psi, or greater than larger, or 30psi than 10 psi, or greater than 60 psi, or greater than 100 psi, or with a large average power than 200 psi, by physically clamping the members to each other or, by directly coupling the surface each other, it is possible to further enhance the thermal contact.

(Via the silver block having a high thermal conductivity and low specific heat capacity, heat is applied to energy) Eppendorf Mastercycler (TM) ep gradient S thermal cycler that such, with respect to block thermal cycler, directly on TCE substrate by placing, it is also possible to increase the heat transfer between the TCE, the substrate in contact with it. Suitable TCE includes a heat pump having a high heating capacity and cooling capacity, and includes a high output of the Peltier device, a (but not limited to). Examples of Peltier devices, 9500/131 / 150B (Ferrotec, Bedford NH), XLT2393 (Marlow, Dallas TX) and the like. When utilized as part of the TCE relates to a thermal cycler of the present invention, the Peltier device is subjected advantageously powered by the H-bridge. Examples of H-bridge devices include 5R7-001 (Oven Industries).

As part of the TCE relates to a thermal cycler of the present invention, when using a Peltier device, using the pulse width modulation about heating and cooling, it is advantageous to power the Peltier device by H-bridge. Temperature feedback from thermosensor to measure the temperature of the sample drives the TCE, to set and maintain the desired sample temperature. Closed-loop temperature control algorithms for control of TCE includes PID control and fuzzy logic control (but not limited to).

Said thermal controller gives the capacity for rapid transitions from one target temperature state to another target temperature state, and a control algorithm. The transition can also be divided into three distinct phases. In phase 1, a large difference between the actual temperature and the target temperature (e.g., 1 to 20 ° C., or greater difference) is. In this phase, the maximum rate (or near) the TCE device, ramping occurs. In Phase 2 (transition phase), the actual temperature and the target temperature approaches more (smaller than approximately 1 to 20 ° C.). In this case, the controller, in order to prevent the overshoot of the solution temperature, with it is necessary to reduce the power of the TCE, with minimal departure and vibration, it is necessary to enable rapid acquisition of the target temperature. In phase 3, the target temperature is acquired, and the controller, in order to maintain the solution in a narrow range of the vicinity of the target temperature, thereby suppressing power against thermal cycler. Temperature measurement using a sensor, as well as provide accurate feedback from the actual temperature, making it possible to overdrive the temperature of the surface temperature of the TCE. Each of the three phases described above, further, it is also possible to subdivided into a number of sub-phases. Thus, faster response time, more accurate temperature control, increased stability, and is given increased resistance to external variations.

In one embodiment, by placing a thin thermocouple into the channel of the substrate surface, it is possible to measure the temperature of the substrate. In other embodiments, the second thermosensor having about the same material as the substrate being utilized, "the distance from the TCE in the second thermosensor is, the reaction chamber on the substrate in contact with the TCE as substantially the same distance from the TCE in, can be accommodated within range "for holding the second thermosensor. Generally, such a second thermosensor, be separated from the substrate (i.e., be isolated type sensor) are also possible. Further, the second thermosensor, can be placed next to the substrate on the first surface of the TCE.

The heat sink may optionally, in order to control the temperature of the heat sink, a variable speed cooling fan, and / or may also comprise a second heating element. In this case, each of the additional element in the sink, and communicates with the second control element. Thereby, it becomes possible to adjust the cooling efficiency of the heat sink. In particular, the temperature of the heat sink, regardless of changes in environmental temperature, it is possible to maintain substantially constant. Also, the heater in advance, it is also possible to adjust the temperature of the heat sink substantially operating temperature.

To promote thermal coupling of the reaction solution in the substrate and TCE, when the thermal cycler is operating, by applying a force for fixing the substrate to the substrate, in contact with the first surface of the TCE and has a thermal conductivity of the contact surface of the substrate can be kept constant. Such forces are preferably means for maintaining the substrate only temporarily for the first surface of the TCE, by the possible means for easily removed when the heat cycle is completed, the application . For example, on top of the first surface of the TCE, it is also possible to arrange the chip compression element (CCE). This makes it possible to place the substrate in between. Then, the chip compression element, be used to hold the substrate in position during operation of the thermal cycler, and can be released to allow the removal of the substrate. CCE, by appropriately integrating the TCE and the heat sink, the CCE, it is possible to improve the thermal coupling between these three members.

Some of CCE in contact with the substrate, can be formed from a low thermal mass insulating material. Such materials include, for example, WF71 Rohcell foam (Inspec foams, Magnolia, AR) foam (bubbles), such as (but not limited to). With the embodiment shown here, the Rohcell preferred. This is the specific heat capacity (or less thermal mass) of 1.4 to 1.6 (J / gK), and has the following thermal conductivity of 0.0345W / mK.

Biochip compression element, one or more clamps, spring, compressible foam, or it possible to inflate to provide a force for holding the substrate on the first surface of TCE, pressure It contains been alveoli (but not limited to). Preferably, the chip compression element, to the surface of the substrate, of from about 5 to about 250 psia, providing a substantially uniform force. More preferably, in order to hold the substrate to the first surface of the TCE, about 20 to about 50 psia, providing a substantially uniform force. Especially, thermal bonding solution (e.g., thermal grease or glycol, etc.) in the absence of, it is possible to provide thermal conduction between the contact surface and the TCE of the biochip substrate. If necessary, it is possible to utilize the thermal coupling solution.

Biochip compression element, the biochip - thermoelectric cooler - empower heatsink. This force, good thermal contact, and which by functions to secure the heat transfer between the uppermost surface of the biochip and TCE.

In one embodiment, insulator low thermal mass, a bladder, is utilized to provide a low thermal mass and insulation properties. In another embodiment, the low thermal mass of the insulator is a foam pad. The clamping force, by a pneumatic cylinder it is possible to give a foam pad, or pneumatically or compressed from bladder, it is possible to provide a closed-cell foam pads. In the latter case, alveoli is, give both insulation and compressive forces.

As described above, the thermal cycler, the first surface of the TCE surface, about four to one hundred fifty ° C. (preferably every second, about 8 to 150 ° C. / sec, more preferably, from about 10 to 150 ° C. / sec ) it is possible to have heating and / or cooling rate of. Thermal cycler, also in solution in the reaction chamber in the substrate in a uniform heat conduction state between the first surface of the TCE (e.g. biochip), about 4-150 ° C. per second (preferably, about 8-150 ° C. / sec, and more preferably, it may have a heating or cooling rate of about 10 to 150 ° C. / sec). Further, the thermal cycler of the present invention, + / - 1.0 ℃ (preferably, + / - 0.50 ° C., more preferably, + / - 0.25 ℃) can have a temperature stability of is there.

Biochip according to another embodiment of the biochip present invention (i.e., a substrate for use with a thermal cycler of the present invention) embodiment of the purposes of illustration, is shown in Figure 1B. The biochip has sixteen microfluidic system, each of which has an inlet and an outlet in fluid communication with each reaction chamber formed within the biochip. However, such disclosure is not meant to be limiting. Rather, there will be to easily recognize by those skilled in the art, the biochip is possible to provide a microfluidic system (described later) of the other quantities, biochips having one system, and, 2 there is also a biochip having more than three systems. The term "plurality" as used herein, two or more, 4 or more, 8 or more, 16 or more, 32 or more, 48 or more, 64 or more, 96 or more, 128 or more, Alternatively, 2 to 16, 2 to 32 carbon atoms, 2 to 48 carbon atoms, 2 to 64 carbon atoms, 2 to 96 carbon atoms, from 2 to 128 pieces, 8 to 128 carbon atoms, 8 to 64 carbon atoms, or 8 to 32 amino microfluidic which means the channel.

Biochips can have a substrate layer and a cover layer. Here, one or more microfluidic systems has grooves and / or recesses are patterned on the substrate layer. To provide fluid access to the microfluidic channel and the reaction chamber, the cover layer within a series of vias (i.e., through-holes and / or the inlet or outlet) can be formed. Also, the series of vias can be placed in any position with respect to the biochip. Further, in order to obtain the same functionality, the vias can also be formed in the substrate layer rather than the cover layer. To complete the microfluidic system, the top surface of the substrate layer, it is possible to bind to the bottom surface of the cover layer. Techniques for producing the polymer-based microfluidic systems, by Becker and Gartner (Becker, 2000, Electrophoresis 21, 12-26 and Becker, 2008, Electrophoresis 390, 89), being reviewed extensively. These, by reference, the entirety of which is incorporated herein. For biochip, unsaturated, partially unsaturated, or saturated cyclic olefin copolymer "COC", unsaturated, partially unsaturated, or saturated cyclic olefin polymers "COP", poly (methyl) methacrylate "PMMA", polycarbonate "PC", polypropylene "PP", polyethylene "PE", polyetheretherketone "PEEK", poly (dimethylsiloxane) "PDMA", using a material such as polyimide "PI" it is possible to manufacture. It is important to choose a plastic having a glass transition temperature higher than the maximum temperature utilized in the amplification reaction. To produce biochips described herein, it is possible to use any number in these processes and materials. In particular, a biochip, a plastic substrate (e.g., sold under the trade names of COC or COP-based polymer (currently, Topas (TM), Zeonex (TM), Zeonor (TM), and Apel (trade mark))) by injection molding, it is possible to produce. In this production procedure, the mold insert and the injection-molding die consisting negative of the structure to be formed is produced by machining and subsequent surface polishing. Mold and insert are both together makes it possible to produce the substrate layer, the substrate is formed, makes it possible to include channel, the reaction chamber structure and vias. The substrate and the cover layer, by applying heat and pressure, it is possible to diffusion bonding.

Alternatively, using a master mold in negative of the structure to be produced, by processing heat embossing a thin thermoplastic film, it is also possible to manufacture a biochip. This master mold, by using the electroforming method for reproducing device that is formed in the solid substrate, can be produced. The substrate of the solid, may be a glass sheet. This sheet is by standard photolithography and chemical etching methods known to those skilled in the art, is patterned. Substrate and the cover layer, by being applied heat and pressure, are diffusion-bonded.

The substrate and the cover layer of the biochip can be constructed from a variety of plastic substrates. The plastic substrate is polyethylene, poly (acrylates) (for example, poly (methyl methacrylate)), poly (carbonate), and, unsaturated, partially unsaturated, or saturated cyclic olefin polymers (COP), Alternatively, unsaturated, partially unsaturated, or contain a saturated cyclic olefin copolymer (COC) (but not limited to). The thickness of the plastic substrate and the cover layer utilized in the process, in order to minimize its mass is maintained thin. Thus, the heat transfer during use between the reaction solution held in a thermal cycler and each reaction chamber is maximized. A plastic substrate and the cover layer are each, independently, less than 2 mm, less than 1 mm, less than 750 [mu] m, less than 650 .mu.m, less than 500 [mu] m, less than 400 [mu] m, less than 300 [mu] m, it is possible to have less than 200 [mu] m, or less than 100μm thick. Alternatively, a plastic substrate and the cover layer are each, independently, 25~2000μm, 25~1000,25~750μm, 25~650mm, 25~500μm, 25~400μm, 25~300μm, 25~200μm, of 25~100μm it is possible with a plastic having a thickness range. Preferably, at least one of the substrate and the cover layer has a thickness of less than about 200 [mu] m. Thus, heat transfer to the reaction solution held in the reaction chamber of the biochip is maximized. More preferably the contact surface of the biochip (surface in contact with the first surface of the TCE) has a thickness of less than about 200 [mu] m.

For each reaction chamber, it is possible to form, for example, as having a 100μL volume less than. Preferably, each reaction chamber is less than about 50 [mu] L, or less than about 40 [mu] L, or less than about 30 [mu] L, or less than about 25 [mu] L, or less than about 20 [mu] L, or less than about 15 [mu] L, or less than about 10 [mu] L, or, less than about 5 [mu] L, or less than about 1 [mu] L, or has a volume of less than about 0.1 [mu] L. Alternatively, each reaction chamber, so as to have a volume in the range from about 0.1μL to about 100 [mu] L, can be formed. Preferably, each reaction chamber is from about 0.1μL to about 10 [mu] L, or has a volume ranging from about 10 [mu] L to about 50 [mu] L. The reaction chamber is generally not coated with a polymer or a silane coating. The reaction chamber can also be designed to have an inlet channel and an outlet channel. Alternatively, for inlet and outlet, it is also possible to use a single channel.

Biochip design of the present invention utilizes the advantages of microfluidic. This advantage, that it has a high surface to volume ratio and shortened diffusion time to maximize heat transfer, and, that it has a uniform heating and cooling include. Use of microfluidic technology, furthermore, provides advantages in terms of fully integrated forensic analysis instruments. Moreover, various layers (e.g., COC layer) to couple, without the use of adhesives, are tested biochip manufactured by diffusion bonding, the biochip, to meet the requirements in the desired application by eliminated, it has been demonstrated that withstand pressures from 100 to 1500 psi. For example, the biochip of the present invention withstands 450 psi. This is sufficient for the desired thermal cycling applications.

It should be noted that certain embodiments of the biochip of the present invention described above, substantially for heating and / or cooling the reaction chamber, nothing these heating elements integrated into the biochip It does not have. Thermal cycling of the reaction chamber in the biochip, externally, e.g., by a thermal cycler of the present invention are given. However, with respect to the biochip of the present invention, it is possible to integrate the heating element.

During operation, a portion of the biochip, it is possible to receive one or more of the reaction solution. Each reaction solution, independently, through one or more inlets in fluid communication with one or more reaction chambers which are formed in the biochip, one or more reagents (e.g., for PCR and a reagent) and / or diffusion samples. By injecting each nucleic acid sample separate separation reaction chamber, it is possible to perform simultaneous amplification of a plurality of samples. To allow multiple samples amplified simultaneously, the injector for simultaneously injecting a plurality of samples to a plurality of sample wells or a buffer well, it is also possible to provide a biochip. Such injection may, for example, one sample in a plurality of samples and provides a single reaction chamber in a plurality of reaction chambers. According to any method known to those skilled in the art, for example, through a needle or tube or channel connecting the sample to the reaction chamber, an electrophoretic transport, by a pneumatic drive, or liquid driven injector, the sample it is possible to introduce into the channel.

Amplification (and, optionally, extraction and quantification of nucleic acid) followed for the production of separation and STR profiles of fragments, via one or more outlet ports in fluid communication with the reaction chamber, the amplification has been a nucleic acid product (relative to e.g. Genebench-FX (TM) 100) can be passed.

Low cost plastic molding of the relatively, the biochip of the present invention makes it possible to those of the disposable. Thus, the work is eliminated that is required for reuse of biochips, also substantially eliminates the possibility of contamination. Discarding used with one-time, for the analysis of low copy number, in that the possibility of contamination (pollution other than the first sample collection) does not exist, it would be particularly advantageous. In situations where both the contamination and work is not in the main considerations, it is also possible to use a reusable plastic and glass biochips.

By utilizing an integrated manner microfluidic, it is possible to manufacture a configuration for executing more than one function in a single biochip. These features, nucleic acid extraction, nucleic acid purification, prior to nucleic acid purification PCR, purification after PCR, purification before sequencing, sequencing, purification after sequencing, nucleic acid separation, nucleic acid detection, reverse transcription, reverse transcription prior to purification, purification after reverse transcription, nucleic acid ligation, and may include hybridization and quantification of nucleic acids. To enable sequential process of the sample, more than one of these functions, it is possible to microfluidic connected. This binding is referred to as the integrated (integration).

By inserting a purification media between the input and output channels, it is possible to obtain one form in a microfluidic DNA extraction. This purification media, as well as can be assumed silica fiber-based, for dissolving the biological sample, to expose the DNA, and, in order to bind the DNA to the purification media, reagents chaotropic salt it is possible to use. The lysate to bind to DNA, via the input channel, is conveyed through the purification media. Bound DNA in order to remove contaminants, it is cleaned by the ethanol-based buffer. For this, via the input channel, by flowing the wash reagent through purification membrane can be achieved. Then, by flowing an appropriate low salt buffer (see, e.g., U.S. Pat. No. 5,234,809 the (Boom)), bound DNA is eluted from the membrane, via an input channel, through the purification membrane Te, it goes out from the output channel.

One approach to DNA quantification in a microfluidic format is based on real-time PCR. In this quantification method, the reaction chamber is formed between the input and output channels. The reaction chamber is attached to the thermal cycler, the optical excitation and detection system, bound to the reaction chamber. Thus, it is possible to measure the fluorescence emission from the reaction solution. The amount of DNA in the sample is correlated with the fluorescence emission intensity from the reaction chamber for each cycle (e.g., Heid et al, Genome Research 1996, 6, see 986-994).

Micro For further information about the integral in the fluid format, the application and the same date, filed by "INTEGRATED NUCLEIC ACID ANALYSIS (integrated analysis of nucleic acids)," entitled U.S. patent application (Attorney Docket No. 07 -801-US), which is incorporated herein by reference. Incidentally, by reference, all of this application is incorporated herein. For further information about the separation and detection in the microfluidic format, this application and was filed on the same date, "Plastic Microfluidic Separation and Detection Platforms (separation platform relates to a microfluidic plastic and detection)" entitled US patent see the application (Attorney Docket No. 07-865-US). Incidentally, by reference, all of this application is incorporated herein.

Microfluidic drive of the present invention is a means for conveying the fluid in the reaction chamber in the integrated bio-chip. One type of microfluidic drive is achieved by built-in membrane pumps. The diaphragm pump, by sequentially applying a positive pressure and negative pressure to the membrane, to convey the fluid. Alternatively, it is also possible to connect the positive displacement pump to the input portion of the microfluidic chamber. Displacement of the pump, forcing the fluid through the microfluidic channel.

Integration is to gate the flow of the fluid in the bio-chip, it is possible to utilize a microfluidic valve. For valve adjustment, utilizing passive structure or active structure, it is possible to realize. Passive valving structure stops the flow of fluid by utilizing capillary pressure, and a capillary valve. Fluid, by receiving the pressure of sufficient magnitude to overcome the capillary force, it is possible to flow through the capillary valve structure. Active valve structure includes a membrane valve, which in the position between the two channels, using a soft (or semi-rigid) structure. By applying pressure against the film, the film closes the channel. When exerting a vacuum on the film, the film is lifted from the channel. This allows passage of fluid.

The amplification method still further aspect, the present invention provides a plurality of nucleic acid loci in one or more target nucleic acids, a method for amplifying simultaneously by rapid polymerase chain reaction (PCR). Such methods are one or more of the reaction solution, comprising the step of providing one or more reaction chambers. Then, each reaction solution, (i) at least at least one copy of one target nucleic acid (the target nucleic acid are the same and or different and are further each target nucleic acid is independently to be amplified and a plurality of loci), (ii) 1 one or more buffers, (iii) 1 one or more salts, (iv) a corresponding set of primers to a plurality of loci to be amplified, (v ) a nucleic acid polymerase, and has a (vi) nucleotides.

Each of the reaction solution, for example, is, respectively, respectively of the target nucleic acid, if necessary, may be the same or different. Thus, for example, multiple simultaneous analysis in the same nucleic acid sample is performed, or multiple nucleic acid samples are processed simultaneously.

Each reaction chamber, it is also possible to hold in the biochip of the present invention as described above, or it can be kept thin in the reaction tube. Thin reaction tubes, it preferably has a wall thickness of less than about 200 [mu] m. Possible, the thin-walled reaction tube, it preferably has a wall thickness of less than about 100 [mu] m.

Primers for PCR amplification, the locus of the target DNA was specifically designed to cross an oligonucleotide sequence. These primers serve as starting points for polymerase extension. To facilitate the analysis of the amplified fragment in a PCR reaction, it is also possible to use labeled primers. Labeled primers are oligonucleotide sequences. These sequences are linked to a detectable moiety. Non-limiting examples are fluorescent dyes. If the PCR is performed using the labeled primers by fluorescence, amplicons with fluorescent labels is generated. Method for performing a fast PCR, it is possible to correspond to both of the primer that is not the labeled primers and labeling. Also, high-speed multiplex PCR has been demonstrated.

Set of primers, for the amplification of multiple loci with target nucleic acid as described above, it is possible to any of those known to those skilled in the art. For example, primers useful in the amplification of one or more loci in the human nucleic acid sample, U.S. Patent No. 5,582,989, U.S. Pat. No. 5,843,660, U.S. Pat. No. 6,221,598, U.S. Pat. No. 6,479,235, U.S. Patent No. 6,531,282 No., and U.S. Patent No. 7,008,771, and U.S. Patent application Publication No. 2003/0180724, No. 2003/0186272, and are described in No. 2004/0137504. Each of these, by reference, are incorporated herein.

Further, primers useful in the amplification of one or more loci in the viral nucleic acid sample is, for example, U.S. Pat. No. 7,312,036, U.S. Pat. No. 6,958,210, U.S. Pat. No. 6,849,407, U.S. Patent No. 6,790,952, and, It is described in U.S. Patent No. 6,472,155. Each of these, by reference, are incorporated herein.

Examples of primers useful in the amplification of one or more loci in a bacterial nucleic acid samples, U.S. Patent No. 7,326,779, U.S. Pat. No. 7,205,111, U.S. Pat. No. 7,074,599, U.S. Pat. No. 7,074,598, U.S. Patent No. 6,664,080 Nos, and it is described in U.S. Patent No. 5,994,066. Each of these, by reference, are incorporated herein.

Salts and buffers includes a well known to those skilled in the art, in honor, it includes those comprising MgCl _2, and Tris-HCl and KCl. Buffers, such as surfactants (e.g. Tweens), dimethyl sulfoxide (DMSO), glycerol, bovine serum albumin (BSA) and polyethylene glycol (PEG), and other well known to those skilled in the art, such as it is also possible to include additives. Nucleotides are, generally, e.g., deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), such as deoxyguanosine triphosphate (dGTP), and deoxythymidine triphosphate (dTTP), deoxyribonucleic a nucleoside triphosphate, also for synthetic material, an amount suitable for amplification of nucleic acid targets are added.

The first period which is suitable for hot start activation of a nucleic acid polymerase, the solution, optionally, it is also possible to heat and held at the first temperature. Generally, the first period is shorter than about 90 seconds. The first temperature, it is also possible to from about 95 ° C. to about 99 ° C.. The polymerase with a possible hot start mechanism to activate within 60 seconds, which utilizes a hot start via antibodies, and also include those utilizing hot start mechanism through aptamer. Alternatively, in the present invention, it is not necessary to use a hot start polymerase.

Thereafter, the temperature of the reaction solution, degenerative conditions, during the annealing conditions and the extended state, for a predetermined circulation number, circulates continuously. Generally, one or more of the reaction solution, at a first cooling rate, is cooled in the annealing state from a denatured state. The first cooling rate is from about 1 to about 0.99 ° C. / sec, or from about 1 to about 100 ° C. / sec, or from about 1 to about 80 ° C. / sec, or from about 1 to about 60 ° C. / sec, or from about 1 to about 40 ° C. / sec, or from about 1 to about 30 ° C. / sec, or from about 1 to about 20 ° C. / sec, about 4 to about 0.99 ° C. / sec, or from about 4 to about 100 ° C. / sec, or from about 4 to about 80 ° C. / sec, or from about 4 to about 60 ° C. / sec, or from about 4 to about 40 ° C. / sec, or from about 4 to about 30 ° C. / sec or about 4 about 20 ° C. / sec, or from about 10 to about 0.99 ° C. / sec, or from about 10 to about 100 ° C. / sec, or from about 10 to about 80 ° C. / sec, or from about 10 to about 60 ° C. / sec, or from about 10 to about 40 ° C. / sec, or from about 10 to about 30 ° C. / sec, Rui is from about 10 to about 20 ℃ / sec. One or more of the reaction solution, at a first heating rate, it is possible to heat from the annealing state to an extended state. The first heating rate is from about 1 to about 0.99 ° C. / sec, or from about 1 to about 100 ° C. / sec, or from about 1 to about 80 ° C. / sec, or from about 1 to about 60 ° C. / sec or, from about 1 to about 40 ° C. / sec, about 1 to about 30 ° C. / sec, about 1 to about 20 ° C. / sec from 4 to about 0.99 ° C. / sec, or from about 4 to about 100 ° C. / sec or about 4 about 80 ° C. / sec, or from about 4 to about 60 ° C. / sec, or from about 4 to about 40 ° C. / sec, about 4 to about 30 ° C. / sec, about 4 to about 20 ° C. / sec or, about 10 to about 0.99 ° C. / sec, or from about 10 to about 100 ° C. / sec, or from about 10 to about 80 ° C. / sec, or from about 10 to about 60 ° C. / sec, or from about 10 to about 40 ° C. / sec, or from about 10 to about 30 ° C. / sec, or from about 10 to about 20 ° C. / sec. And / or, one or more of the reaction solution, at a second heating rate, is heated from the extended state to denatured state. This and the second heating rate from about 1 to about 0.99 ° C. / sec, or from about 1 to about 100 ° C. / sec, or from about 1 to about 80 ° C. / sec, or from about 1 to about 60 ° C. / sec , or from about 1 to about 40 ° C. / sec, about 1 to about 30 ° C. / sec, about 1 to about 20 ° C. / sec, about 4 to about 0.99 ° C. / sec, or from about 4 to about 100 ° C. / sec, or from about 4 to about 80 ° C. / sec, or from about 4 to about 60 ° C. / sec, or from about 4 to about 40 ° C. / sec, about 4 to about 30 ° C. / sec, about 4 to about 20 ° C. / sec , or from about 10 to about 0.99 ° C. / sec or, alternatively from about 10 to about 100 ° C. / sec, or from about 10 to about 80 ° C. / sec, or from about 10 to about 60 ° C. / sec, from about 10 about 40 ° C. / sec, or from about 10 to about 30 ° C. / sec, or from about 10 to about 20 ° C. / Byodea . Finally, the reaction solution is, for providing one or more of the amplified spread product, is held in the final state.

Degenerative conditions, generally in a time range from about 1 to 30 seconds, there is a possibility that over the range of about 90 to 99 ° C.. The actual time and temperature, the enzyme is dependent on the primer and target. For amplifying human genomic DNA, with respect to the Applied Biosystems (AB) multiplex STR kits, preferably about 95 ° C. for about 5 seconds.

Temperature and time of annealing, affects the specificity and the ability of the primer which is coupled to a plurality of loci in target nucleic acid, it is particularly important for multiplex PCR reactions. In the annealing step, by properly combining the complete set of primer pairs, multiplex amplification of a plurality of loci (e.g., having a PHR and internal loci intensity signal balance acceptable, one or more full STR profile) it is possible to generate. With respect to specific primer pairs, annealing condition, the range of about 50 ° C. to 70 ° C., and is likely over time ranging from about 1 second to 30 seconds. The actual time and temperature, the enzyme is dependent on the primer and target. For the AB multiplexed STR kit for amplifying human genomic DNA, preferably about 59 ° C. for 15 seconds.

Temperature and time of extension are those which affect primarily allele product yield, it is an inherent characteristic of the enzyme under study. Incidentally, extension rate as reported by the manufacturer is noted that most of the case, given for single reaction. Elongation rate for multiplex reaction is likely to be much slower. For certain enzymes, extended state in the range of about 60 ° C. to 75 ° C., and is likely over time ranging from about 1 second to 30 seconds. The actual time and temperatures are dependent enzymes, primers, and the target. For the AB multiplexed STR kit for amplifying human genomic DNA, preferably about 72 ° C. for about 5 seconds. Preferably, with respect to continue a predetermined number of cycles, the reaction solution is in the third speed, is heated from the extended state to denatured state. This third speed is from about 1 to about 0.99 ° C. / sec, or from about 1 to about 100 ° C. / sec, or from about 1 to about 80 ° C. / sec, or from about 1 to about 60 ° C. / sec or, from about 1 to about 40 ° C. / sec, or from about 1 to about 30 ° C. / sec, or from about 1 to about 20 ° C. / sec from 4 to about 0.99 ° C. / sec, or from about 4 to about 100 ° C. / sec , or from about 4 to about 80 ° C. / sec, or from about 4 to about 60 ° C. / sec, or from about 4 to about 40 ° C. / sec, or from about 4 to about 30 ° C. / sec, or from about 4 about 20 ° C. / sec, or from about 10 to about 0.99 ° C. / sec, or from about 10 to about 100 ° C. / sec, or from about 10 to about 80 ° C. / sec, or from about 10 to about 60 ° C. / sec, or from about 10 to about 40 ° C. / sec, or from about 10 to about 30 ° C. / sec, there have , From about 10 to about 20 ° C. / sec. Generally, a predetermined number of cycles is selected to be a cycle of about 10 to 50 times. However, if necessary, it is also possible to use fewer cycles (or more).

The final extension time until incomplete NTA begins to increase, it is possible to greatly reduced. For certain enzymes, the final extension temperature, may ranging from about 60 ° C. to 75 ° C., the time range may span from about 0 seconds to 300 seconds. The actual time and temperature, the enzyme is dependent on the primer and target. For the AB multiplexed STR kit for amplifying human genomic DNA, preferably about 72 ° C. for about 90 seconds.

In addition to the 3-step thermal cycle approach described above, this process is further suitable for two-step thermal cycle approach. In this approach, the reaction solution is between the denatured state and annealing / extension state, for a predetermined number of cycles, is continuously circulated. This approach utilizes a primer is designed to anneal at the extension temperature. Thus, annealing and extension steps, it is possible to share the same temperature. The reduction of the temperature transition rate, the cycle time can be further shortened.

In certain embodiments, it is possible from about 5 to about 20 minutes, to obtain a plurality of amplified nucleic acid products. In another particular embodiment, about 5 to 10 minutes, from about 1 5 minutes, or in less than 5 minutes, it is possible to obtain a plurality of amplified nucleic acid products. From the target nucleic acid of less than about 10 ng, it is possible to generate respective amplified nucleic acid products. Preferably, the nucleic acid of less than about 5ng or, nucleic acids less than about 2ng or, nucleic acids less than about 1ng or, nucleic acids less than about 0.5 ng, or less than about 0.2ng nucleic Alternatively, about 0. the nucleic acid of less than 1ng or, nucleic acids less than about 0.05ng or, can be a nucleic acid of less than about 0.006 ng, to produce an amplified nucleic acid product.

For example, certain biological warfare agents in clinical samples or environmental samples, or human, bacterial in plants and animals, such as certain viral or fungal infections, in other embodiments, the amplified nucleic acid product and it can be generated from at least one copy of the target nucleic acid. For example, the sample to be analyzed, prior to the multiplex amplification reaction, a copy of less than 1000 in the target nucleic acid (e.g., 1 to 1000 copies), 400 fewer than copying, 200 fewer than copies, less than 100 copy, fewer than 50 copies, 30 fewer than copying, less than 10 copies, needs to include a copy of less than one.

Furthermore, the gene of the target nucleic acid locus, when present in one or more copies in the genome, those less than a single genome equivalent to DNA, can be used for the amplification is there.

In all of the methods described above, in order to obtain a sufficient amplification of the loci in the target nucleic acid, for a predetermined number of cycles, it is possible to perform the thermal cycle (this is readily determined by those skilled in the art Is possible). For example, a predetermined number of cycles, ranging from about 10 to about 50 cycles, and preferably can range from about 20 50 cycles. Further, in all of the methods described above, at least two loci in one or more nucleic acids can be amplified simultaneously. Depending on the desired application, the more than four, ten from 5, 10 from the 20, 20 from the 30, or 10 from 250 loci are amplified simultaneously. For example, for amplification of STR loci, there are 20 loci may be preferred from the 10.

Preferably, the temperature of the reaction solution is circulated by a thermal cycler of the present invention (described above). By compensating the delayed response of the PCR solution, it is also possible to use a commercially available block thermal cycler for rapid thermal cycling. This compensation is to set a higher block temperature than the desired solution temperature in the heating step, and is done by, for setting the desired lower block temperature than the solution temperature in the cooling step. However, this mode of operation, since it is necessary to determine the temperature setpoint that is required to compensate for the slow ramping reaction empirically, difficult implementation. Further, the block, since the feedback and control is executed subsequently, and, in order to monitor the dissolution temperature is not conducted, the profile of the repetitive (repeatability) and reproducibility (Reproducibility) is, external factors ( there is a possibility that is affected by including the change in room temperature). Therefore, it is impossible to reproduce the temperature profile of the solution.
Using the approaches described herein, a number of polymerases are commercially available, can be applied rapidly PCR applications. Generally, the nucleic acid polymerase has an elongation rate of at least 100 bases / second. Thermus aquaticus (Taq), Pyrococcus furiosus (Pfu), Pyrococcus Wosei (Pwo), Thermus flavus (TfI), Thermus thermophilus (Tth), Thermus Ritorisu (TIi) and Thermotoga - Maritime (Tma numerous polymerases including) is useful for PCR amplification. These enzymes, improved versions of these enzymes, and, the combinations of these enzymes, Roche, Invitrogen, Qiagen, commercially available from vendors including Strategene, and Applied Biosystems. Typical enzymes, PHUSION (New England Biolabs, Ipswich, MA), Hot MasterTaq (TM) (Eppendorf), PHUSION Mpx (Finnzymes), PyroStart (Fermentas), KOD (EMD Biosciences), Z-Taq (TAKARA), and includes CS3AC / LA (KlenTaq, University City, MO) to. Enzymes which are widely used with respect to PCR amplification for STR typing is Taq polymerase. Moreover, it improved the TaqGold is, Identifiler (TM), Profiler (TM), and shipped with COfiler (TM) kit.

Although not the generation of a complete profile required, in certain embodiments, PCR conditions are presented here, it can be generated from human target nucleic acid with high efficiency, a full STR profile is there. Full profile for STR autosomal, for example, amelogenin, D8S1179, D21S11, D7S820, CFS1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818, FGA or more of these, , it is possible to have loci, such as the other STR loci including mini STR and Y-STR analysis. Criteria for optimization in protocol, generates a complete profile, it is signal strength, the dynamic range, internal locus signal strength balance, PHR, incomplete NTA, include stutter, and the total cycle time.

According to one embodiment, the protocol utilizes a thermal cycler SpeedSTAR enzyme and the present invention is to produce a full STR profile, the total cycle time for biochip reactions and tube reaction, respectively, 17.3 minutes and it is possible to shorten the 19.1 min. In this protocol, approximately 98 ° C. denaturing conditions of about 4 seconds, annealing condition is about 59 ° C. for about 15 seconds, extended state is about 72 ° C. for about 7 seconds, then the final state of about 90 about 70 ° C. in seconds.

In certain embodiments, at least 10 times, 20 times or 30 times the total cycle time for multiplex PCR cycles, it can range from about 1 minute to about 90 minutes. Preferably, at least 10 times, the total cycle time is about 20 times or 30 times the multiplex PCR cycles, from about 1 minute to about 90 minutes, or from about 1 minute to about 85 minutes, or from about 1 minute to about 80 minutes , or from about 1 minute to about 75 minutes, or from about 1 minute to about 70 minutes, or from about 1 minute to about 65 minutes, or about 60 minutes to about 1 minute, or from about 1 minute to about 55 minutes , or from about 1 minute to about 50 minutes, or from about 1 minute to about 45 minutes, or from about 1 minute to about 40 minutes, or from about 1 minute to about 35 minutes, or from about 1 minute to about 30 minutes , or from about 1 minute to about 25 minutes, or from about 1 minute to about 20 minutes, or from about 1 minute to about 15 minutes, or from about 1 minute to about 10 During or in the range of from about 1 minute to about 5 minutes. In other embodiments, at least 10 times, 20 times or 30 times the total cycle time for multiplex PCR cycles, less than about 90 minutes. Preferably, at least 10 times, 20 times or 30 times the total cycle time for multiplex PCR cycles, less than about 89 minutes, less than about 85 minutes, less than about 80 minutes, less than about 75 minutes, less than about 70 minutes, about 65 less minutes, less than about 60 minutes, less than about 55 minutes, less than about 50 minutes, less than about 45 minutes, less than about 40 minutes, less than about 35 minutes, less than about 30 minutes, less than about 25 minutes, less than about 20 minutes, about 15 less minutes, less than about 10 minutes, less than about 5 minutes, less than about 4 minutes, less than about 3 minutes, less than about 2 minutes, or less than about 1 minute.

The present invention, for performing multiplex PCR amplification of multiple loci in a nucleic acid sample, and, at the same biochip platform, for executing a manufacturing and / or analysis methods according to at least one other sample, with one or more microfluidic systems are intended integrated biochip. For example, within the microfluidic system in biochips, each of which has a flow direction toward the outlet port from the inlet port, the system is, it can comprise a plurality of reaction chambers, the plurality of reaction chambers the first reaction chamber in the, whereas that through inlet port in fluid communication with, the final reaction chamber in a plurality of reaction chambers, in fluid outlet port in fluid communication with, furthermore, the system is continuous in the reaction chamber are fluidly connected along the flow direction for each pair, it is possible to provide at least one microchannel. At least one reaction chamber in each microfluidic system can be less than 200μm from the contact surface of the biochip substrate. Thus, for performing multiplex PCR, the thermal conductivity of the thermal cycler of the present invention it is promoted in the reaction chamber.

In each microfluidic system of the biochip, for each of the remaining reaction chambers, nucleic acid extraction, nucleic acid purification, nucleic hybridization, nucleic acid ligation, prior to nucleic acid purification PCR, purification after PCR, purification before sequencing, sequencing, purification after sequencing, separation and detection, reverse transcription, purification prior to reverse transcription, and / or purification after reverse transcription, electrophoretic separation, it is possible to adapt to the nucleic acid detection. The term "purification" as used herein, are likely to interfere with any of the reaction chamber process listed above, refers to the removal of the reaction components. The reaction components, anionic, cationic, oligonucleotides, nucleotides, preservatives, include enzymes, or inhibitors.

Embodiments described below, certain embodiments of the present invention, and illustrate the various uses. These is a than only been described for illustrative purposes and are not to be understood as limiting the present invention.

Example Example 1
Rapidly custom thermal cycler and microfluidic biochip PCR reaction solution temperature, controllably, and in order to perform high-speed cycling by being able to reproducibly heating and cooling, the thermal cycler of the present invention as shown in FIG. 1A used. This instrument, receiving a microfluidic biochip 16 chamber, comprising a high output thermoelectric cooler / heater that is attached to a high performance heat sink. 16 each PCR reaction solution were placed in individual chambers of the microfluidic biochip that bind to the heat pump by applying a compression pressure 0.2MPa using a clamp mechanism. Figure 1B shows a photograph of disposable plastic microfluidic biochip 16 samples. Each PCR chamber depth 500 [mu] m, a width of about 1 mm, containing the PCR reaction solution 7 [mu] l.

In the following examples the measurement and temperature profiles, Eppendorf Mastercyler (TM) ep gradient S (Eppendorf North America, Westbury, NY) were performed using any amplification reaction tubes. Using a thermocouple sensor types K of diameter 127μm attached directly to the block to obtain a block temperature profile of the instrument. For the reaction solution profile, a thermocouple sensor types K of diameter 127 [mu] m, was placed on a reaction solution 20μL in thin PCR tubes. To acquire data at 100Hz of speed, it was carried out data collection in the Omega HH506RA Multilogger thermometer set.

Using a thermal cycler of Example 1 having a plastic biochips 16 samples as a reactor, it was amplified reactions in biochips. By inserting a thermocouple into the detection chamber of the biochip was monitored temperature of the solution in a microfluidic biochip.

PCR- reaction mixture components and cycling conditions 9947A genomic DNA (Promega, Madison, WI) the AmpFlSTR as template (TM) Profiler Plus (registered trademark) IDPCR Amplification Kit (Profiler Plus ID kit) (Applied Biosystems, Foster City, CA It was multiplex PCR reaction). Were used for amplification polymerase, Profiler Plus ID AmpliTaq Gold which kit being supplied to the (R) DNA polymerase (TaqGold (TM)) or other polymerases, namely, SpeedSTAR HS DNA Polymerase (SpeedSTAR) (Takara BIO USA Inc., Madison, WI), KOD Hot Start DNA polymerase (KOD) (EMD Biosciences Inc., Gibbstown, NJ), or PyroStart (TM) Fast PCR Master Mix (PyroStart) (Fermentas Inc., Glen Burnie, MD) of It was either. A combination of a polymerase specific buffer and deoxynucleotide triphosphates, using the labeled multiplex primer from Profiler Plus ID kit was performed multiplex PCR using other polymerases. Use Eppendorf Mastercyler (TM) ep gradient S, thin PCR tubes 0.2mL (Eppendorf North America, Westbury, NY) every tube PCR with Been. Use 16 samples of biochips, any biochip reaction was amplified in a thermal cycler of Figure 1A.

It was prepared following PCR reaction mixture was used to thermal cycling.
The reaction of the Standard TaqGold (TM):
Standard TaqGold (TM) multi-reactants, the reaction volume of 25 [mu] L, and Profiler Plus ID reaction mixture 9.55MyuL, and 9947A genomic DNA 1 ng, and Profiler Plus ID primer set of 5 [mu] L, of 2.25U TaqGold (TM ) it was made of. Select cycling conditions (block temperature and time) according to the manufacturer's recommendations, 11 min beginning 95 ° C. (hot start), followed by 1 min at 94 ° C. for 28 cycles (denaturation), 1 minute at 59 ° C. (annealing ), 1 minute at 72 ° C. (extension), was set to 45 minutes at 60 ° C. the final extension.

Optimized TaqGold (TM) reaction:
For optimized TaqGold (TM) reactant fast cycle, the Profiler Plus ID reaction mixture 3.82MyuL, and 9947A genomic DNA 1 ng, and 2μL of Profiler Plus ID primer set, of 0.9 U TaqGold (TM) containing the door, it was carried out in 10μL reaction volume. 95 ° C. In 11 minutes, 10 seconds at 98 ° C. 28 cycles, 45 sec at 59 ° C., 30 sec at 72 ° C., 15 minutes at 72 ° C. The final extension, in the reaction was circulated.

SpeedSTAR tube reaction was:
SpeedSTAR PCR mixture components for tube PCR were as follows. And 2μL of Profiler Plus ID primer set, and 9947A genomic DNA, 1xFast Buffer I (Takara BIO USA Inc., Madison, WI) and a deoxynucleotide triphosphates 200 [mu] M, and the SpeedSTAR of 0.315U, 10 [mu] L of the reaction It was in the volume. Cycling conditions for rapid execution was set below. Following 1 minute (enzyme activation) at 95 ° C., 4 seconds at 98 ° C. 28 cycles, 15 sec at 59 ° C., 5 sec at 72 ° C., was 1 min at 72 ° C. The final extension.

SpeedSTAR biochip reaction was:
For biochip PCR, the reaction mixture 7μL is, contains the Profiler Plus ID primer set 1.4MyuL, and 9947A genomic DNA, and 1xFast Buffer I, and deoxynucleotide triphosphates 200 [mu] M, the SpeedSTAR of 0.42U which was. The cycling parameters, 70 seconds at 95 ° C., 28 cycles 4 seconds 98 ° C., 15 sec at 59 ° C., were set 7 seconds at 72 ° C., a final extension at 1:30 minutes 70 ° C..

KOD reaction was:
It has entered the reaction volume of 10 [mu] L, and 2μL of Profiler Plus ID primer set, 1XKOD buffer (EMD Biosciences Inc., Gibbstown, NJ) and a deoxynucleotide triphosphates 200 [mu] M, and 9947A genomic DNA 1 ng, 1. and 5mM magnesium sulfate (MgSO _4), in the KOD of 0.2 U, was amplified in KOD. Cycling conditions were as follows. Following 2 min at 95 ° C., 4 seconds at 98 ° C. for 28 cycles, 30 sec at 59 ° C., 10 sec at 72 ° C., was 1 min at 72 ° C. At the end of the extension.

PyroStart reaction was:
The reaction mixture in PyroStart in one final concentration also has a 2μL of Profiler Plus ID primer set for the reaction of 10 [mu] L, are those containing a 9947A genomic DNA 1 ng, it was circulated in the following. 1 min at 95 ° C., 4 seconds at 98 ° C. 28 cycles, 20 sec at 59 ° C., followed by 30 seconds at 72 ° C., was performed for 1 minute final extension at 72 ° C..

Multiplex PCR with other STR typing kit:
Using other STR typing kit (AmpFlSTR (TM) Identifiler (TM) (Identifiler) (AmpFlSTR (TM) Cofiler (TM) PCR Amplification Kit (COfiler) (Applied Biosystems)), a full STR profile the suitability of SpeedSTAR for generate, at the reaction conditions for SpeedSTAR in Profiler Plus ID kit as described above, were examined in the tube and the biochip. in these reactions, the Profiler Plus ID primer set, respectively It was replaced from the kit to the primer set.

The reproducibility of studies of repeatability tube and biochips, using 9947A genomic DNA of 1ng as a template was performed using TaqGold (TM) and SpeedSTAR. As reproducibility of tubes were prepared five individual reactants. Each performed three biochip PCR with eight reaction was measured biochip reproducibility.

The study of sensitive SpeedSTAR amplification in sensitive tube and biochip were performed using the following amounts of 9947A template DNA. The tube: 4ng, 2ng, 1.5ng, 1ng, 0.5ng, 0.25ng, 0.125ng, 0.1ng, 0.05ng, 0.03ng, 0.02ng, 0.01ng, 0.006ng; Bio in the chip: 4ng, 2ng, 1.5ng, 1ng, 0.5ng, 0.25ng, 0.1ng, 0.05ng, 0.025ng, 0.02ng, 0.015ng, 0.01ng, 0.006ng. The reaction in each template level, was performed twice.

Measurement of STR of separation and detection
Use Network Biosystem Genebench-FX (TM) Series100, the amplification products were separated and detected (Pyzowski and Tan, Advances in Biochip-Based Analysis: A Rapid Field-Based Approach 59th Annual Meeting of the American Academy of Forensic Sciences San Antonio, TX, February 19-24, 2007). This instrument is especially developed for STR analysis, optimized. Each amplification product until 2.7μL, Hi-Di (R) formamide and 0.1μL of Genescan 500 LIZ internal lane standard (both Applied Biosystems, Foster City, CA) of 10.2μL was added. Denatured for 3 min at 95 ° C., after snap cooled (snap Cooling) on ​​ice, loaded with sample separation chip, by applying an electric field of 90 seconds 350 V / cm, moved electrophoretically to the separation channel . Following this, an electric field of 150 V / cm applied along the separation channel to separate the DNA fragments. All of the separation was carried out at 50 ℃.

Data analysis
GeneMarker (TM) HID STR Human Identification Software, Version 1.51 (SoftGenetics LLC, State College, PA) was used to analyze the data. To standardize the signal strength to internal lane standard, it was determined the percentage of the stutter and incomplete NTA and PHR. By dividing the alleles of a small signal strength locus in a more alleles of a larger signal intensity, it calculates a PHR. By dividing the signal strength of the template fragment (-A) at the signal intensity adenylation fragments (+ A), to calculate the level of incomplete NTA.

Example 2
Thermal cycling instruments and temperature profile commercial thermal using cycler a thin PCR tube, and Example 1 of thermal cycling microfluidic biochips using Klar between the reaction solution of conventional PCR tubes and microfluidic biochips in, it was carried out an amplification reaction. The tube reaction, using Eppendorf Mastercyler (TM). Figure 2A using the conventional STR cycling protocol shows one block and the temperature of the reaction solution of 28 thermal cycles in the tube. The Mastercycler (TM) heating and cooling system is based on a heat pump with an integrated block for inserting the tube. The set value of time and temperature, 1 minute at 98 ° C. for denaturation, 1 minute at 72 ° C. for 1 minute, and extension at 59 ° C. for annealing. Comparing the temperature profile of the thermal block and reaction solution, the delay of the reaction solution temperature are shown for block temperature. Measurements heating and cooling rate of the block is 5.6 ° C. / sec and 4.9 ° C. / sec, measuring heating and cooling rate of the solution is 4.8 ° C. / sec and 3.3 ° C. / sec. Block temperature at 14 seconds extension (72 ° C.) until denaturation (98 ° C.) transitions, but the solution does not reach a temperature of 39 seconds setting. Each block and solution, the transitions between steps of denaturation and annealing (59 ° C.), it takes 10 seconds and 27 seconds, between steps of annealing and extension according 7 seconds and 24 seconds.

The temperature profile of the block and the reaction solution for one of the Eppendorf Mastercyler (TM) of 28 thermal cycles at high speed cycling conditions shown in Figure 2B. The set value of time and temperature, 5 sec at 98 ° C. for denaturation, 15 seconds at 59 ° C. for annealing, and 5 seconds at 72 ° C. to elongation. However, the reaction solution was suppressed with a delay, the solution does not reach a temperature of a desired set point.

The temperature profile of the heat pump and the reaction solution for one of the 28 thermal cycles of the thermal cycler of the present invention using fast cycling conditions were also measured (Fig. 3). The measurement of the reaction solution temperature was used to detect the chamber within the biochip. The set value of time and temperature, 4 seconds 95 ° C. for denaturation, 15 seconds at 59 ° C. for annealing, and 7 seconds at 72 ° C. to elongation. Measurements heating and cooling rate of the heat pump is 21.5 ° C. / sec and 21.7 ° C. / sec, measuring heating and cooling rate of the reaction solution is 14.8 ° C. / sec and 15.4 ° C. / sec.

Thus, thermal cycler of the present invention can be rapidly heated cooling the reaction solution in commercial block-based 3 to 5 times faster than cycler. Extension at the heat pump, denaturation, and annealing the transition time between steps of, 1.7 seconds, 2.1 seconds, and 0.7 seconds, 2.7 seconds in the solution, 4.5 seconds and 2.2 in seconds. The thermal cycler of the present invention, the reaction solution can reach about 7 times rapidly required temperature than block-based cycler, as a result, a predetermined controlled incubation temperature and time, high speed cycling conditions .

Example 3
Measurement multiple polymerase PCR enzyme tubes were measured potential using a fast multi-STR analysis, based in part on hot start activation time and extension rate, and selecting a candidate substance. The reported characteristics of the four polymerase selected as experimental evaluation as compared to the recommended conditions of TaqGold (TM), shown in Table 1 (A).

Measured enzyme has an extension rates that are reported to vary from about 15 to 200 nucleotides / sec. In general, the reported elongation rate is based on singlet amplification may be somewhat smaller as multiple applications.

In order to obtain a full STR profile in the time of minimal, the PCR conditions tubes were initially investigated for these four enzymes. In all reactions, the use of buffers and enzyme concentrations primer pair with suppliers from Profiler Plus ID kit as recommended, human genomic DNA of 1ng amplifies, the profile resulting, Genebench-FX (TM) Series It separated using 100 detects and determines the size and quantified. Denaturation, annealing, and various time elongation steps and the temperature were measured, together with the STR signal strength suitable translation varying from 71.7 minutes 19.13 minutes TaqGold (TM) in SpeedSTAR, for PCR amplification It showed total time of Table 1 (B)]. The conditions of the process of the present invention, it is possible to rapidly perform the 2-10 fold amplification than recommended conditions TaqGold (TM).

Additional measurements of enzymes for forensic related with performance and signal strength includes the stutter and incomplete NTA and PHR levels of Table 1 (B)]. All enzymes, using the Profiler Plus ID primer, it is possible to perform advanced multiplex amplification. SpeedSTAR, Pyrostart, signal strength for the KOD and optimized TaqGold (TM) All reactions, or is substantially the same as those generated using the PCR conditions standard TaqGold (TM), or raised by there.
Respect incomplete NTA, both SpeedSTAR and PyroStart, as well as optimized TaqGold (TM) reaction showed 3-fold higher levels at the maximum than the standard TaqGold (TM) reaction. Most alleles, the level is below 15% of the translated acceptable limits. As described below, these alleles with incomplete high levels NTA, can be reduced to less than 15% of the translated acceptable limits. KOD polymerase has a 3'-5 'exonuclease activity, it does not generate a fragment with a A-overhang. Therefore, all alleles was only one nucleotide shorter than these alleles ladder counterpart (allelic ladder counterpart).

Optimized TaqGold (TM), the relative level of stutter observed in SpeedSTAR and PyroStart reaction is similar to the range of stutter produced by standard TaqGold (TM) reaction. That is, the range of stutter produced by KOD is slightly higher than the stutter standard TaqGold (TM).

Example 4a
Based on the rapid PCR protocol above results using SpeedSTAR polymerase tube and biochip, the total cycle time to minimize the total STR profile satisfying the signal strength, PHR, incomplete NTA, and stutter translation requirements for the purpose of achieving, it was selected for measurement of further biochips the SpeedSTAR polymerase.

Using SpeedSTAR in thermal cycler microfluidic 16- samples of the biochip of the present invention, the set value of the time and temperature for the amplification, 70 seconds at 95 ° C. in a hot-start activation, followed by 28 cycles, 4 seconds at 95 ° C. for denaturation, 15 seconds at 59 ° C. for annealing, a 7 sec at 72 ° C. to elongation. Between 17.3 min all protocols times, performing final extension for 90 seconds at 72 ° C.. The tube reaction Eppendorf Mastercycler, carried 19.13 minutes, which is 1 minute at 95 ° C., 4 seconds at 98 ° C. to 28 cycles, 15 sec at 59 ° C., and followed by five seconds at 72 ° C., final extension the comprised the block time and temperature are set to 1 minute initial activation time at 72 ° C..

4A and 4B, by using the DNA of 0.5 ng, at 7μL biochip (FIG. 4A) and 10μL of the tube reaction (Figure 4B), showed a STR profile generated by SpeedSTAR cycling conditions described above, Table 2, using a standard protocol, shows the signal intensities of all the Profiler Plus ID allele from TaqGold (TM) reaction and tubes SpeedSTAR biochip and tube. Signal intensity of the reaction of SpeedSTAR biochip 0.5ng is that the approximately 2-fold higher on average than in the case of the reaction of 1ng standard TaqGold (TM), the signal intensity of the reaction of SpeedSTAR tube 0.5ng is substantially the same on average as in the reaction of TaqGold (TM).

Example 4b
Using SpeedSTAR polymerase tube and biochip, the allele characterization Example 4a fast PCR to characterize the products of high-speed PCR reactions, PHR, the number of incomplete NTA and stutter was performed. Biochip and tube reaction using SpeedSTAR polymerase, compared to the reaction of TaqGold (TM), a large imbalance in peak height between loci. PHR alleles generated in biochip reaction is 0.70 to 0.95, is substantially the same in the tube. In other words, all is within the tolerance of the translation guidelines. Reaction using SpeedSTAR is, PHR is about 10% less than that measured in a standard TaqGold (TM) reaction. Similarly, most of the incomplete level of NTA alleles in the reaction of both the biochip and tube using SpeedSTAR is approximately the same (2.0 and 10.6%). That is, both approximately two times greater than TaqGold (TM) control reactions. Exceptions are incomplete NTA of D3S1358 alleles, which, TaqGold (TM) 4.8 from 7 times greater With SpeedSTAR than used. That is, even in this case, the level of incomplete NTA is 12% or less in SpeedSTAR enzyme. Finally, the level of stutter of the reaction based on both biochip and tube using SpeedSTAR is about 6.0 to 14.1%, about on average than the level of the tube reaction standard TaqGold (TM) 1.6 times larger.

Microfluidic biochip reactions using template DNA of 0.5ng the standard TaqGold (TM) using the template of 1ng produce about 2-fold greater signal strength than that of the reaction. This result is to suggest, TaqGold (TM) enzyme SpeedSTAR enzymes and conventional reaction biochip is that act at the same efficiency. Ie, DNA concentration of the biochip is about 1.8 times the tube, this corresponds to that the signal strength is 2 times greater. In contrast, fast reaction tube base control TaqGold (TM) less efficient than the reaction. In other words, the product yield was reduced by approximately 40%, presumably the result of poor cycling profile, this occurs when the commercial thermal cycler was used in a high speed thermal cycling. This even in the environment, the signal strength, the translation is well beyond the level required for (data not shown) can be significantly increased by extending the only extension time several seconds per cycle. Repeatability and reproducibility of the signal strength of the high-speed PCR reactions biochip and tube are similar to those of conventional reactions.

Gene signal strength allele loci for the reaction of fast biochip and tube, as compared to TaqGold (TM) reaction shows a high imbalance levels. Balance of the signal strength of the loci are primer concentration, annealing temperature and time, and is affected by a number of factors including the molecular weight of the locus. STR amplification kit for use in these experiments, with optimized set of primer concentrations were for TaqGold (TM) enzyme and recommended cycling protocol. By adjusting primer concentrations to be used in the amplification reaction, it is possible to modify the signal strength of the locus (Henegariu et al., Biotechniques 1997, 23, 504-11).

The relationship between the signal intensity and the template level fast response biochip and tube expected, the signal intensity increases at generally the template. Good peak Mofo Logistics is observed at high template level 4ng at all alleles (which produces allele having a signal strength exceeding the 12000RFU). 0.03ng in the following template level, lack of part of the allele occurs. Amplification reaction is carried out in a limited number of template DNA strand in solution, in the case leading to stochastic amplification, this effect is observed (Walsh et al., PCR Methods Appl. 1992, 1, 241-250). If there is immediately detectable signal both alleles alleles and small signal strength of greater signal strength in the template level of 0.006 ng, biochips are combined in the separation and detection of Genebench-FX (TM) Series 100 and it shows a high sensitivity for fast reactions of the tube, showing the utility of this system to a low copy number analysis. Taken together, this data is also rapid PCR methods and thermal cycler of the present invention, as well as Genebench measurements, suggesting that it has a dynamic range of high template.

Example 4c
Characterized biochip of DNA template level and alleles in rapid PCR reactions (Figure 5A) and tubes the effect of the template DNA for the signal strength of the high-speed PCR reaction using SpeedSTAR polymerase in reaction (Figure 5B), Figures 5A and 5B to show. Allele selected for analysis is amelogenin, alleles, STR profile and FGA23 and 24 has a maximum signal level arrangement D7S820 10 and 11, alleles minimum signal for the profile It was equipped with a level. In the reaction of both SpeedSTAR biochip and tube, as DNA template level increases from 0.006ng to 4 ng, the signal intensities of all the alleles is increased. In template level 0.006 ng, it was observed signal strength at amelogenin peak of 58RFU of 111RFU and tube reaction biochip. In template level 4 ng, signal strength 12680RFU is in biochip, 5570RFU was observed in the reaction tube. All alleles observed in both reaction types, showed good peak Mofo Logistics.

In fast biochip reaction (Fig. 6A), PHR is 0.6-1.0, as a template level changes from 0.05ng to 4.0 ng. The 0.05ng following template levels, Examples of missing allele occurs, when the PHR of 0 is observed, PHR is reduced to 0.025 ng. Similar results, although they are generally exhibits somewhat less PHR than biochip reaction (Fig. 6B), is observed in the reaction of the high-speed tubes. The biochip reaction, the level of incomplete NTA is 15% or less as following template level 2.0 ng. The tube reaction, the level of incomplete NTA is greater than 15% template level 1 ng, are dramatically 4ng increased.

Two major differences between reaction of the biochip and the tube is the relative concentration of the temperature profile and the template and the polymerase reaction solution. Level of incomplete NTA decreases as more polymerases is available. In biochip reaction, show experimental data is beyond the range of DNA template 4.0ng from 0.5 ng, as SpeedSTAR polymerase content increases from 0.3U to 1.2 U, incomplete NTA level There is that reduced about 50% (FIGS. 7A and 7B). Level of stutter fast reaction biochip and tube is a relatively constant, beyond the range of the template level 4.0ng from 0.25 ng, typically 15% was less in all alleles (Figures 8A and 8B ).

If the reaction itself generates data to be legal subjects that meet the forensic translation guidelines, STR amplification reaction rate is only related. The FBI has a general guideline for use for STR translation, individual laboratories, have set limits to be met before it can be considered profile is acceptable based on the confirmation work (Holt et al. , J. Forensic Sci 2002, 47 (1), p.66-96;.. LaFountain et, J. Forensic Sci, 2001, 46 (5), 1191-8).

Condition indicated herein, it is possible to generate a high-speed STR profiles that meet these guidelines. PHR of 0.5ng of template in the reaction of the biochip and tube are stated to be required 0.6 or more levels, and satisfies the translation guidelines consistent with results reported previously (Leclair et al, J. Forensic Sci. 2004, 49, 968-80). The large amount of DNA template, PHR is remains relatively constant, less than that of TaqGold (TM) reaction of 1 ng. At low copy number, the PHR is dominated due to stochastic fluctuations by amplification.

Level of incomplete NTA is based on the ability of the polymerase to fully adenylation all STR amplification products. In the conventional amplification, by attaching a "pigtail" to the primer, by extending the final extension time, to achieve this. Incomplete level of NTA reaction 0.5ng biochip and tube as described herein are within the translation guidelines.

Level of incomplete NTA increases with increasing DNA template (as a result of the ratio of DNA to polymerase has increased), polymerase amount, by increasing extension time per cycle, and a final extension time, reducing be able to. The latter two methods is that they in order to increase the reaction time, not well suited for high-speed multiplex amplification. Increasing the polymerase concentration is effective, with a fast PCR compatible. Stutter is the result of displacement of DNA strands in elongation (Walsh et al., Nucleic Acids Res. 1996, 24 (14), 2807-12). Level of stutter reaction 0.5ng biochip and tube as described herein is in the translation guidelines are consistent with reported previously listed. As expected, the level of stutter is as unrelated to the DNA template level.

Example 4d
A study of the repeatability and reproducibility
The repeatability and reproducibility of the reaction of the fast biochip using SpeedSTAR polymerase (Table 3A) and tube (Table 3B), do the same PCR reaction 24 with three PCR biochip, performs five same tube reaction It was calculated by. The biochip reactions confidence value of the signal intensity (CV) varies from 17 to 24%, and a tube reaction varies from 15 to 34%. CV in standard TaqGold (TM) reaction was 6% to 21%.

Compared to the range of 5% to that observed in standard TaqGold (TM) reaction of 10%, CV of PHR is 14% at most biochip, is 21% in the tube reaction. CV incomplete NTA in biochip reaction was 6% to 28%, which is 2-13% in the tube reaction. Again, these changes are similar to the range of 4 observed in the reaction of the standard TaqGold (TM) 28%. CV of stutter in biochip is 9% from 4, 6% from the 2 in the tube, are similar with the range 4 to 13% of the observed in standard TaqGold (TM) reaction.

Example 4e
Using other commercially available compatible above the same fast biochip and tube conditions the STR kits, were calculated set of samples using a primer set from COfiler (TM) and Identifiler (TM) kit. 9A and 9B, a thermal cycler of the present invention as described herein, SpeedSTAR enzymes, and using these primer sets using the protocol indicates the achievement of all profiles. Each, are equally suitable for these commercial kits. Despite all profile was achieved, it was observed imbalance signal intensity over locus.

Example 5
Fast Sequencing thermal cycling instruments and methodologies using a thermal cycler can be applied to high-speed DNA sequencing reactions. Fast thermal cycler to achieve in this way, the measurement and biochips are identical to those used in PCR. The sequencing, applying different reaction solution, polymerase and cycling temperature. The current commercial sequencing reactions, (in GE Amersham DYEnamic (TM) ET Terminato Cycle Sequencing Kit) 49 minutes, and 2.25 hours (at AB Big Dye V3.1) according. By utilizing NetBio thermal cycler with conventional reagents, it was possible to shorten the sequencing reaction time to 21 minutes.

Use biochip comprising thermocycler and 16 lanes disclosed herein, we have achieved high-speed sequencing. The final reaction volume in the chip was 7 [mu] L. The sequencing reactions half strength, according to the manufacturer's protocol, was set by GE Healthcare of DYEnamic (TM) ET Terminator Cycle Sequencing Kit. Therefore, in order to accommodate a final volume of 7 [mu] L, it was reduced every volume. The reaction template was Bacillus subtilis 0.1pmol with fragment sizes of 343bp (B. subtilus).

(95 ° C. for 20 seconds, 15 seconds at 50 ° C., and 60 ° C. 60 seconds) and the first protocol consisting of 30 cycles of (total cycle time is 51.7 minutes), 5 sec at 95 ° C., 50 ° C. for 15 seconds, and a second protocol consisting of 60 ° C. for 30 seconds 30 cycles (total cycle time is 29 minutes), 5 sec at 95 ° C., 10 sec at 50 ° C., and 20 sec at 60 ° C. 30 cycles of (total cycle time is 21.6 minutes) by the third protocol consisting demonstrated three cycling protocol. Each sequencing reaction was purified by precipitated ethanol and separated into Genebench FX Series 100. Average PHRED score sequencing PCR products 343bp for three cycling protocol, respectively, 282,287, and was 279. That is, in less than 22 minutes in the chip, it is possible to prove the sequencing of the product of 343 bp. Figure 10 shows the DNA sequence of the high-speed DNA sequencing protocols.

Overall, using the systems and methods described herein, the multiplex amplification of one or more nucleic acid biochips based, was amplified in a very short total reaction time for the reaction run in a thin tube nucleic acid It provides product, has the advantage of utilizing thermal cycling unit currently commercialized.

Claims (73)

1. In the thermal cycler,
   Comprises a temperature control element (TCE),
   The first surface of the TCE is a sample chamber holding a solution, and is configured to receive a detection chamber that holds the thermosensor,
   The thermo sensor, in order to set or maintain the solution on the desired temperature, giving feedback to the TCE, thermal cycler.
2. In the thermal cycler,
   Comprises a temperature control element (TCE),
   The first surface of the TCE is a sample chamber holding a solution, and is configured to receive a detection chamber that holds the first thermosensor,
   The thermo sensor, in order to set or maintain the solution on the desired temperature, and give feedback to the TCE,
   The first surface, further arranged to monitor the temperature of the in the TCE first surface, and a second thermosensor, a thermal cycler.
3. In the thermal cycler,
   Comprises a temperature control element (TCE), a first surface of the TCE is is configured to receive a sample chamber, wherein the chamber holds the solution and thermosensor, the thermo sensor , in order to set or maintain the solution on the desired temperature, giving feedback to the TCE, thermal cycler.
4. Wherein comprises a first chip compression element disposed on a surface (CCE) further in TCE, thereby, to allow insertion of a substrate between the CCE and the TCE, the CCE is the TCE and the substrate giving thermal conduction between thermal cycler according to claim 1, 2 or 3.
5. The CCE will contain a low thermal mass insulating material, a thermal cycler according to claim 4.
6. The CCE contains a foam pad, a thermal cycler according to claim 4.
7. The CCE is contains one or a plurality of clips, thermal cycler according to claim 4.
8. The CCE contains a bladder thermal cycler of claim 4.
9. The first surface of the TCE is configured to receive a thin-walled tube, the thermal cycler according to claim 1 or 2 or 3.
10. About from 4 to 150 ° C. per second, and it has a heating and cooling rate in the first surface of the TCE, thermal cycler according to claim 1 or 2 or 3.
11. Further comprising a heat sink, the heat sink has a variable speed cooling fan to control the temperature of the heat sink, the thermal cycler according to claim 1 or 2 or 3.
12. It said heat sink further comprises a second thermocycler for controlling the temperature of the heat sink, the thermal cycler according to claim 11.
13. In place of the sample in the substrate that the first surface and the heat conduction in the TCE, + / - 1.0 and has a temperature stability of ° C., thermal cycler according to claim 1 or 2 or 3 .
14. The TCE is the heat pump has a high heating and cooling capacity or, a Peltier device with high output, the thermal cycler according to claim 1 or 2 or 3.
15. The heat sink is a copper-based fan-cooled heat sink having a cooling fan, a thermal cycler according to claim 11.
16. It said heat sink has the following thermal resistance approximately 0.4 ° C. / W, the thermal cycler of claim 15.
17. In the system,
    Includes a biochip having one or more reaction chambers,
    Each reaction chamber has the outflow channel of the inlet channel and the microfluidic microfluidic, each reaction chamber, which is less than 200μm from the contact surface of the biochip substrate,
    Further comprising a thermal cycler having a temperature control element (TCE),
    The solution first surface of the TCE is a sample chamber holding a solution, and, to a temperature which is configured to receive a detection chamber that holds the thermosensor, the thermo sensor, as desired to set or maintained, and provide feedback to the TCE,
    The biochip substrate contact surface and the heat conduction to that system.
18. In the system,
    Includes a biochip having one or more reaction chambers,
    Each reaction chamber has the outflow channel of the inlet channel and the microfluidic microfluidic, each reaction chamber, which is less than 100μm from the contact surface of the biochip substrate,
    Further comprising a thermal cycler having a temperature control element (TCE),
    The solution first surface of the TCE is a sample chamber holding a solution, and, to a temperature which is configured to receive a detection chamber that holds the thermosensor, the thermo sensor, as desired to set or maintained, and provide feedback to the TCE,
    Further, the thermal cycler were placed to monitor the temperature of the first surface of the TCE, it has a second thermosensor,
    The biochip substrate contact surface and the heat conduction to that system.
19. The inlet channel of the microfluidic outlet channel of the microfluidic or both of these, have a via of claim 17 or 18 system.
20. The thermal cycler comprises a first chip compression element disposed on a surface (CCE) further in the TCE, thereby, to allow insertion of a substrate between the CCE and the TCE, the CCE is, giving a heat transfer between the TCE and the substrate, the system according to claim 17 or 18.
21. The chip compression element includes a low thermal mass insulating material, A system according to claim 20.
22. The CCE contains a foam pad system of claim 20.
23. The CCE is contains one or a plurality of clips, the system according to claim 20.
24. The CCE contains a celled The system of claim 20.
25. Wherein the contact surface of the biochip substrate, in order to hold to the first surface in thermal contact in the TCE, the CCE has given pressure from about 5 to about 50 psi, according to claim 20 system.
26. The reaction chamber is not covered by the polymer, the silane coating or BSA, the system according to claim 17 or 18.
27. Even when there is no thermal coupling solution, the heat transfer between the biochip contact surface of the substrate and the first surface of the TCE is given, the system according to claim 17 or 18.
28. About from 4 to 150 ° C. per second, and it has a heating and cooling rate in the first surface of the TCE, the system according to claim 17 or 18.
29. The thermal cycler have the ability to be about 4-150 ° C. The heating and cooling rate in the reaction chamber per second, according to claim 17 or 18 system.
30. Further comprising a copper-based fan-cooled heat sink having a cooling fan system according to claim 17 or 18.
31. Approximately 0.4 ° C. / W further comprises a heat sink having the thermal resistance system of claim 17 or 18.
32. Further comprising a heat sink, the heat sink has a variable speed cooling fan to control the temperature of the heat sink according to claim 17 or 18 system.
33. Further comprising a heat sink, the heat sink has a second heating element for controlling the temperature of the heat sink further according to claim 17 or 18 system.
34. The thermal cycler, a temperature stability in the location of the sample within the bio-chip, + / - has the ability to 1.0 ° C., according to claim 17 or 18 system.
35. The TCE is high heating and heat pump having a cooling capacity or, a Peltier device with high output, the system of claim 17 or 18.
36. The biochip substrate, organic materials, inorganic materials, crystalline materials or and an amorphous substance, system of claim 17 or 18,.
37. The biochip substrate contains a plastic material, the system of claim 36.
38. The biochip substrate will contain a cyclic olefin copolymer (COC), according to claim 37 systems.
39. The biochip substrate, and a 8-128 amino microfluidic system The system of claim 17 or 18.
40. Each reaction chamber has a volume of less than about 100 [mu] L, The system of claim 17 or 18.
41. A method for amplifying a plurality of loci in a nucleic acid solution at the same time,
    Includes the step of providing one or more reaction chambers,
    Each reaction chamber,
    (I) a nucleic acid comprising at least one copy of at least one target nucleic acid to be amplified solution,
    (Ii) 1 one or more buffers,
    (Iii) 1 one or more salts,
    (Iv) a corresponding set of primers to each of the plurality of loci to be amplified,
    (V) a nucleic acid polymerase and,
    (Vi) A method has nucleotides, a,
    The temperature of the nucleic acid solution in the reaction chamber, denaturing conditions, among the annealing conditions and the extended state, for a predetermined number of cycles, at a heating and cooling rate of about 4-150 ° C. / sec, continuously thermally cycled is allowed, how Accordingly, within about 97 minutes, including the step of generating a plurality of amplified loci to each reaction chamber.
42. To provide one or more amplified nucleic acid product, it includes the step of maintaining one or more of the reaction solution to a final state, The method according to claim 41.
43. A method for amplifying a plurality of loci in a nucleic acid solution at the same time,
    Includes the step of providing one or more reaction chambers,
    Each reaction chamber,
    (I) a nucleic acid comprising at least one copy of at least one target nucleic acid to be amplified solution,
    (Ii) 1 one or more buffers,
    (Iii) 1 one or more salts,
    (Iv) a corresponding set of primers to each of the plurality of loci to be amplified,
    (V) a nucleic acid polymerase and,
    (Vi) A method has nucleotides, a,
    The temperature of the nucleic acid solution in the reaction chamber, over a predetermined number of cycles, at a heating and cooling rate of about four to one hundred and fifty ° C. / sec, continuously thermally cycled, thereby, within about 97 minutes, the the method comprising the step of generating a plurality of amplified loci in the reaction chamber.
44. A method for simultaneously amplifying five or more loci in a nucleic acid solution,
    Includes the step of providing one or more reaction chambers,
    Each reaction chamber,
    (I) a nucleic acid comprising at least one copy of at least one target nucleic acid to be amplified solution,
    (Ii) 1 one or more buffers,
    (Iii) 1 one or more salts,
    (Iv) 5 or more sets of corresponding primer loci to be amplified,
    (V) a nucleic acid polymerase and,
    (Vi) A method has nucleotides, a,
    The temperature of the nucleic acid solution in the reaction chamber, denaturing conditions, among the annealing conditions and the extended state, for a predetermined number of cycles, at a heating and cooling rate of about 4-150 ° C. / sec, continuously thermally cycled is allowed, thereby, the method comprising the step of generating five or more amplified loci to each reaction chamber.
45. The nucleic acid solution is in the biochip method according to claim 44.
46. The nucleic acid solution is in the thin tube, The method of claim 44.
47. The 5 or more amplified loci is produced in less than about 97 minutes, The method of claim 44.
48. The amplified loci is produced in less than about 45 minutes, The method according to any one of claims 43 to 47.
49. Prior to the continuous thermal cycling,
    One or more of the reaction solution, and heating to a first temperature suitable for hot start activation of a nucleic acid polymerase,
    A step of maintaining said one or more reaction solution, a first period which is suitable for hot start activation of a nucleic acid polymerase, the first temperature,
    Furthermore comprising, A method according to any one of claims 43 to 47 a.
50. The first period is less than about 90 seconds, The method of claim 49.
51. The first temperature is about 90 ° C. to about 99 ° C., The method of claim 49.
52. The thermal cycle is given by the thermal cycler according to claim 1, the method according to any one of claims 43 to 47.
53. It said nucleic acid polymerase, has an elongation rate of at least 100 bp / sec, the method according to any one of claims 43 to 47.
54. Each reaction chamber has a volume of less than about 100 [mu] L, The method according to any one of claims 43 to 47.
55. Each reaction chamber, away from the thermal cycler distance of less than about 200 [mu] m, method according to any one of claims 43 to 47.
56. Each nucleic acid solution contains a copy from about 1 to about 1000 in the target nucleic acid, the method according to any one of claims 43 to 47.
57. It said nucleic acid polymerase, SpeedSTAR, PHUSION, Hot MasterTaq (TM), a PHUSION Mpx, PyroStart, KOD, Z-Taq or CS3AC / LA,, The method according to any one of claims 43 to 47.
58. Analysis of each amplified nucleic acid product is meets guidelines forensic interpretation method according to any one of claims 43 to 47.
59. The modified state is about 95 ° C. for about 4 seconds, The method according to any one of claims 43 to 47.
60. The annealing condition is about 59 ° C. for about 15 seconds, The method according to any one of claims 43 to 47.
61. The extended state is about 72 ° C. for about 7 seconds A method according to any one of claims 43 to 47.
62. The final state is about 70 ° C. for about 90 seconds, The method according to any one of claims 43 to 47.
63. One or more of the reaction solution, at a first cooling rate of about 10 to about 50 ° C. / sec, is cooled to the annealing conditions of a modified state, The method according to any one of claims 43 to 47 .
64. One or more of the reaction solution, at a first heating rate of about 10 to about 50 ° C. / sec, is heated from the annealing state to an extended state, the method according to any one of claims 43 to 47 .
65. One or more of the reaction solution, at a second heating rate from about 10 to about 50 ° C. / sec, is heated from the extended state to denatured state, The method according to any one of claims 43 to 47 .
66. One or more amplified nucleic acid product is obtained within from about 10 to about 90 minutes, The method according to any one of claims 43 to 47.
67. Each reaction solution, from about 0.005 contain target nucleic acids from about 10 ng, the method according to any one of claims 43 to 47.
68. Said target nucleic acid, human nucleic acid, comprising a nucleic acid of microorganisms nucleic or viruses, A method according to any one of claims 43 to 47.
69. 10 from 250 loci are amplified simultaneously, the method according to any one of claims 43 to 47.
70. The locus amelogenin, D8S1179, D21S11, D7S820, CFS1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818, FGA or includes a plurality of these, claim the method according to 69.
71. Wherein the predetermined number of cycles is between about 10 to about 50 cycles, the method according to any one of claims 43 to 47.
72. One or more thin-walled reaction tubes has one or a plurality of reaction chambers, the method according to any one of claims 43 to 47.
73. In integrated biochip system,
    Has a biochip comprising at least two reaction chambers are in fluid micro fluid communication, the first reaction chamber, has a thermal cycler and thermal conductivity,
    The thermal cycler is provided with a temperature control element (TCE), a first surface of the TCE is a sample chamber holding a solution, and, to receive the sensing chamber holding the thermosensor is configured, the thermo sensor, in order to set or maintain the solution on the desired temperature, and give feedback to the TCE,
    Contacting surface of the biochip, it has a first surface and a thermal conduction in a thermal cycler,
    Further, the thermal cycler, and a second reaction chamber connected first reaction chamber and the fluid,
    (I) nucleic acid extraction,
    (Ii) nucleic acid purification,
    (Iii) prior to nucleic acid purification PCR,
    (Iv) purification after PCR,
    (V) sequencing the previous purification,
    (Vi) sequencing,
    (Vii) purification after sequencing,
    (Viii) nucleic acid separation,
    (Ix) nucleic acid detection,
    (X) reverse transcription,
    (Xi) reverse transcription of a previous purification,
    (Xii) clean after reverse transcription,
    (Xiii) nucleic acid ligation,
    (Xiv) nucleic acid hybridization,
    (Xv) has quantified the second reaction chamber suitable for,
    The first reaction chamber, which is less than 200μm from the contact surface of the biochip, integrated biochip system.

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